Karlene Cimprich

All Publications

• AAV-mediated genome editing is influenced by the formation of R-loops. Molecular therapy : the journal of the American Society of Gene Therapy Puzzo, F., Crossley, M. P., Goswami, A., Zhang, F., Pekrun, K., Garzon, J. L., Cimprich, K. A., Kay, M. A. 2024

  Abstract

  Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

  View details for DOI 10.1016/j.ymthe.2024.09.035

  View details for PubMedID 39369271

• HLTF resolves G4s and promotes G4-induced replication fork slowing to maintain genome stability. Molecular cell Bai, G., Endres, T., Kühbacher, U., Mengoli, V., Greer, B. H., Peacock, E. M., Newton, M. D., Stanage, T., Dello Stritto, M. R., Lungu, R., Crossley, M. P., Sathirachinda, A., Cortez, D., Boulton, S. J., Cejka, P., Eichman, B. F., Cimprich, K. A. 2024

  Abstract

  G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.

  View details for DOI 10.1016/j.molcel.2024.07.018

  View details for PubMedID 39142279

• Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids. Nature structural & molecular biology Stoy, H., Zwicky, K., Kuster, D., Lang, K. S., Krietsch, J., Crossley, M. P., Schmid, J. A., Cimprich, K. A., Merrikh, H., Lopes, M. 2023

  Abstract

  Transcription-replication collisions (TRCs) are crucial determinants of genome instability. R-loops were linked to head-on TRCs and proposed to obstruct replication fork progression. The underlying mechanisms, however, remained elusive due to the lack of direct visualization and of non-ambiguous research tools. Here, we ascertained the stability of estrogen-induced R-loops on the human genome, visualized them directly by electron microscopy (EM), and measured R-loop frequency and size at the single-molecule level. Combining EM and immuno-labeling on locus-specific head-on TRCs in bacteria, we observed the frequent accumulation of DNA:RNA hybrids behind replication forks. These post-replicative structures are linked to fork slowing and reversal across conflict regions and are distinct from physiological DNA:RNA hybrids at Okazaki fragments. Comet assays on nascent DNA revealed a marked delay in nascent DNA maturation in multiple conditions previously linked to R-loop accumulation. Altogether, our findings suggest that TRC-associated replication interference entails transactions that follow initial R-loop bypass by the replication fork.

  View details for DOI 10.1038/s41594-023-00928-6

  View details for PubMedID 36864174

• R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response. Nature Crossley, M. P., Song, C., Bocek, M. J., Choi, J., Kousorous, J., Sathirachinda, A., Lin, C., Brickner, J. R., Bai, G., Lans, H., Vermeulen, W., Abu-Remaileh, M., Cimprich, K. A. 2022

  Abstract

  R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1(refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the patternrecognition receptors cGAS and TLR3(ref.8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2(ref.9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.

  View details for DOI 10.1038/s41586-022-05545-9

  View details for PubMedID 36544021

• ATR activity controls stem cell quiescence via the cyclin F-SCF complex. Proceedings of the National Academy of Sciences of the United States of America Salvi, J. S., Kang, J., Kim, S., Colville, A. J., de Morree, A., Billeskov, T. B., Larsen, M. C., Kanugovi, A., van Velthoven, C. T., Cimprich, K. A., Rando, T. A. 2022; 119 (18): e2115638119

  Abstract

  SignificanceThe replication stress response protein, ATR, is active in quiescent muscle stem cells in response to DNA:RNA hybrids, and this activity maintains quiescence by ensuring degradation of key cell-cycle transition factors by the E3 ubiquitin ligase cyclin F-SCF complex. This is critical for understanding how stem cells regulate a state of prolonged and reversible cell-cycle arrest and how genome integrity is maintained over time. Together, these studies offer a unique picture of a molecular mechanism controlling stem cell quiescence.

  View details for DOI 10.1073/pnas.2115638119

  View details for PubMedID 35476521

• Walking a tightrope: The complex balancing act of R-loops in genome stability. Molecular cell Brickner, J. R., Garzon, J. L., Cimprich, K. A. 2022

  Abstract

  Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic.

  View details for DOI 10.1016/j.molcel.2022.04.014

  View details for PubMedID 35508167

• Quantitative DNA-RNA Immunoprecipitation Sequencing with Spike-Ins. Methods in molecular biology (Clifton, N.J.) Crossley, M. P., Cimprich, K. A. 2022; 2528: 381-410

  Abstract

  R-loops are three-stranded nucleic acid structures, comprising an RNA-DNA hybrid and a displaced strand of ssDNA. R-loops have important physiological roles in cells, but deregulation of R-loop dynamics can also have harmful cellular outcomes. The genome-wide mapping of R-loops offers an unbiased approach to study R-loop biology in a wide range of contexts. Here we present a protocol to sequence RNA-DNA hybrids genome-wide with strand-specificity and high resolution. We also include information on how to prepare and incorporate into the workflow appropriate internal spike-in standards which facilitate accurate normalization of the sequencing signal, thereby providing quantitative insights into R-loop formation between different experimental samples.

  View details for DOI 10.1007/978-1-0716-2477-7_26

  View details for PubMedID 35704206

• Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging. The Journal of cell biology Crossley, M. P., Brickner, J. R., Song, C., Zar, S. M., Maw, S. S., Chédin, F., Tsai, M. S., Cimprich, K. A. 2021; 220 (9)

  Abstract

  R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.

  View details for DOI 10.1083/jcb.202101092

  View details for PubMedID 34232287

• HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis. Molecular cell Bai, G. n., Kermi, C. n., Stoy, H. n., Schiltz, C. J., Bacal, J. n., Zaino, A. M., Hadden, M. K., Eichman, B. F., Lopes, M. n., Cimprich, K. A. 2020

  Abstract

  DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.

  View details for DOI 10.1016/j.molcel.2020.04.031

  View details for PubMedID 32442397

• qDRIP: a method to quantitatively assess RNA-DNA hybrid formation genome-wide. Nucleic acids research Crossley, M. P., Bocek, M. J., Hamperl, S. n., Swigut, T. n., Cimprich, K. A. 2020

  Abstract

  R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA-DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA-RNA immunoprecipitation (qDRIP), a method combining synthetic RNA-DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA-DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA-DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.

  View details for DOI 10.1093/nar/gkaa500

  View details for PubMedID 32544226

• R-Loops as Cellular Regulators and Genomic Threats. Molecular cell Crossley, M. P., Bocek, M., Cimprich, K. A. 2019; 73 (3): 398–411

  Abstract

  During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide.

  View details for PubMedID 30735654

• An intrinsic S/G2 checkpoint enforced by ATR. Science (New York, N.Y.) Saldivar, J. C., Hamperl, S., Bocek, M. J., Chung, M., Bass, T. E., Cisneros-Soberanis, F., Samejima, K., Xie, L., Paulson, J. R., Earnshaw, W. C., Cortez, D., Meyer, T., Cimprich, K. A. 2018; 361 (6404): 806–10

  Abstract

  The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

  View details for PubMedID 30139873

• Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates. Cell systems Daigh, L. H., Liu, C., Chung, M., Cimprich, K. A., Meyer, T. 2018

  Abstract

  Faithful DNA replication is challenged by stalling of replication forks during Sphase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed Sphase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout Sphase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged Sphase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout Sphase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.

  View details for PubMedID 29909278

• Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses CELL Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T., Cimprich, K. A. 2017; 170 (4): 774-+

  Abstract

  Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

  View details for PubMedID 28802045

  View details for PubMedCentralID PMC5570545

• The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nature reviews. Molecular cell biology Saldivar, J. C., Cortez, D. n., Cimprich, K. A. 2017; 18 (10): 622–36

  Abstract

  One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.

  View details for PubMedID 28811666

• Conflict Resolution in the Genome: How Transcription and Replication Make It Work CELL Hamperl, S., Cimprich, K. A. 2016; 167 (6): 1455-1467

  Abstract

  The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes.

  View details for DOI 10.1016/j.cell.2016.09.053

  View details for Web of Science ID 000389470500010

  View details for PubMedID 27912056

  View details for PubMedCentralID PMC5141617

• Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nature methods Hess, G. T., Frésard, L., Han, K., Lee, C. H., Li, A., Cimprich, K. A., Montgomery, S. B., Bassik, M. C. 2016

  Abstract

  Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.

  View details for DOI 10.1038/nmeth.4038

  View details for PubMedID 27798611

• Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage. eLife Stork, C. T., Bocek, M., Crossley, M. P., Sollier, J., Sanz, L. A., Chédin, F., Swigut, T., Cimprich, K. A. 2016; 5

  Abstract

  The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

  View details for DOI 10.7554/eLife.17548

  View details for PubMedID 27552054

• Breaking bad: R-loops and genome integrity. Trends in cell biology Sollier, J., Cimprich, K. A. 2015; 25 (9): 514-522

  Abstract

  R-loops, nucleic acid structures consisting of an RNA-DNA hybrid and displaced single-stranded (ss) DNA, are ubiquitous in organisms from bacteria to mammals. First described in bacteria where they initiate DNA replication, it now appears that R-loops regulate diverse cellular processes such as gene expression, immunoglobulin (Ig) class switching, and DNA repair. Changes in R-loop regulation induce DNA damage and genome instability, and recently it was shown that R-loops are associated with neurodegenerative disorders. We discuss recent developments in the field; in particular, the regulation and effects of R-loops in cells, their effect on genomic and epigenomic stability, and their potential contribution to the origin of diseases including cancer and neurodegenerative disorders.

  View details for DOI 10.1016/j.tcb.2015.05.003

  View details for PubMedID 26045257

  View details for PubMedCentralID PMC4554970

• HLTF's Ancient HIRAN Domain Binds 3' DNA Ends to Drive Replication Fork Reversal. Molecular cell Kile, A. C., Chavez, D. A., Bacal, J., Eldirany, S., Korzhnev, D. M., Bezsonova, I., Eichman, B. F., Cimprich, K. A. 2015; 58 (6): 1090-1100

  Abstract

  Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3' ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro through its interaction with 3' ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell.

  View details for DOI 10.1016/j.molcel.2015.05.013

  View details for PubMedID 26051180

  View details for PubMedCentralID PMC4475461

• Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability. Molecular cell Sollier, J., Stork, C. T., García-Rubio, M. L., Paulsen, R. D., Aguilera, A., Cimprich, K. A. 2014; 56 (6): 777-785

  Abstract

  R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.

  View details for DOI 10.1016/j.molcel.2014.10.020

  View details for PubMedID 25435140

  View details for PubMedCentralID PMC4272638

• Causes and consequences of replication stress NATURE CELL BIOLOGY Zeman, M. K., Cimprich, K. A. 2014; 16 (1): 2-9

  Abstract

  Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.

  View details for DOI 10.1038/ncb2897

  View details for Web of Science ID 000329042000003

• NEK8 Links the ATR-Regulated Replication Stress Response and S Phase CDK Activity to Renal Ciliopathies. Molecular cell Choi, H. J., Lin, J., Vannier, J., Slaats, G. G., Kile, A. C., Paulsen, R. D., Manning, D. K., Beier, D. R., Giles, R. H., Boulton, S. J., Cimprich, K. A. 2013; 51 (4): 423-439

  Abstract

  Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.

  View details for DOI 10.1016/j.molcel.2013.08.006

  View details for PubMedID 23973373

• ATR phosphorylates SMARCAL1 to prevent replication fork collapse GENES & DEVELOPMENT Couch, F. B., Bansbach, C. E., Driscoll, R., Luzwick, J. W., Glick, G. G., Betous, R., Carroll, C. M., Jung, S. Y., Qin, J., Cimprich, K. A., Cortez, D. 2013; 27 (14): 1610-1623

  Abstract

  The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.

  View details for DOI 10.1101/gad.214080.113

  View details for Web of Science ID 000322011500008

  View details for PubMedID 23873943

  View details for PubMedCentralID PMC3731549

• A Role for the MRN Complex in ATR Activation via TOPBP1 Recruitment MOLECULAR CELL Duursma, A. M., Driscoll, R., Elias, J. E., Cimprich, K. A. 2013; 50 (1): 116-122

  Abstract

  The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.

  View details for DOI 10.1016/j.molcel.2013.03.006

  View details for Web of Science ID 000317558300012

  View details for PubMedID 23582259

  View details for PubMedCentralID PMC3669687

• SHPRH and HLTF Act in a Damage-Specific Manner to Coordinate Different Forms of Postreplication Repair and Prevent Mutagenesis MOLECULAR CELL Lin, J., Zeman, M. K., Chen, J., Yee, M., Cimprich, K. A. 2011; 42 (2): 237-249

  Abstract

  Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis.

  View details for DOI 10.1016/j.molcel.2011.02.026

  View details for Web of Science ID 000289873700011

  View details for PubMedID 21396873

  View details for PubMedCentralID PMC3080461

• Continued primer synthesis at stalled replication forks contributes to checkpoint activation JOURNAL OF CELL BIOLOGY Van, C., Yan, S., Michael, W. M., Waga, S., Cimprich, K. A. 2010; 189 (2): 233-246

  Abstract

  Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer-template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

  View details for DOI 10.1083/jcb.200909105

  View details for PubMedID 20385778

• A Genome-wide siRNA Screen Reveals Diverse Cellular Processes and Pathways that Mediate Genome Stability MOLECULAR CELL Paulsen, R. D., Soni, D. V., Wollman, R., Hahn, A. T., Yee, M., Guan, A., Hesley, J. A., Miller, S. C., Cromwell, E. F., Solow-Cordero, D. E., Meyer, T., Cimprich, K. A. 2009; 35 (2): 228-239

  Abstract

  Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

  View details for DOI 10.1016/j.molcel.2009.06.021

  View details for Web of Science ID 000268643700011

  View details for PubMedID 19647519

  View details for PubMedCentralID PMC2772893

• The structural determinants of checkpoint activation GENES & DEVELOPMENT MacDougall, C. A., Byun, T. S., Van, C., Yee, M., Cimprich, K. A. 2007; 21 (8): 898-903

  Abstract

  Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5'- and 3'-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage.

  View details for DOI 10.1101/gad.1522607

  View details for Web of Science ID 000245743900003

  View details for PubMedID 17437996

  View details for PubMedCentralID PMC1847708

• Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint GENES & DEVELOPMENT Byun, T. S., Pacek, M., Yee, M. C., Walter, J. C., Cimprich, K. A. 2005; 19 (9): 1040-1052

  Abstract

  The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

  View details for DOI 10.1101/gad.1301205

  View details for Web of Science ID 000228984200006

  View details for PubMedID 15833913

  View details for PubMedCentralID PMC1091739

• A novel protein activity mediates DNA binding of an ATR-ATRIP complex JOURNAL OF BIOLOGICAL CHEMISTRY Bomgarden, R. D., Yean, D., Yee, M. C., Cimprich, K. A. 2004; 279 (14): 13346-13353

  Abstract

  The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

  View details for DOI 10.1074/jbc.M311098200

  View details for Web of Science ID 000220478500011

  View details for PubMedID 14724280

• ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation CURRENT BIOLOGY Barr, S. M., Leung, C. G., Chang, E. E., Cimprich, K. A. 2003; 13 (12): 1047-1051

  Abstract

  Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. Here, we used a microscopy-based approach to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. Our data show that in undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage.

  View details for DOI 10.1016/S0960-9822(03)00376-2

  View details for Web of Science ID 000183588000024

  View details for PubMedID 12814551

• A requirement for replication in activation of the ATR-dependent DNA damage checkpoint GENES & DEVELOPMENT Lupardus, P. J., Byun, T., Yee, M. C., Hekmat-Nejad, M., Cimprich, K. A. 2002; 16 (18): 2327-2332

  Abstract

  Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.

  View details for DOI 10.1101/gad.1013502

  View details for Web of Science ID 000178129600003

  View details for PubMedID 12231621

  View details for PubMedCentralID PMC187437

• AAV-mediated genome editing is influenced by the formation of R-loops. bioRxiv : the preprint server for biology Puzzo, F., Crossley, M. P., Goswami, A., Zhang, F., Pekrun, K., Garzon, J. L., Cimprich, K. A., Kay, M. A. 2024

  Abstract

  Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

  View details for DOI 10.1101/2024.05.07.592855

  View details for PubMedID 38766176

• The crosstalk between DNA repair and immune responses MOLECULAR CELL Cimprich, K. A., Li, G., Demaria, S., Gekara, N. O., Zha, S., Chen, Q. 2023; 83 (20): 3582-3587

  Abstract

  In recent years, increasing evidence has highlighted the profound connection between DNA damage repair and the activation of immune responses. We spoke with researchers about their mechanistic interplays and the implications for cancer and other diseases.

  View details for Web of Science ID 001106798600001

  View details for PubMedID 37863025

• Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination-deficient cancer models. The Journal of clinical investigation Mehibel, M., Xu, Y., Li, C. G., Moon, E. J., Thakkar, K. N., Diep, A. N., Kim, R. K., Bloomstein, J. D., Xiao, Y., Bacal, J., Saldivar, J. C., Le, Q., Cimprich, K. A., Rankin, E. B., Giaccia, A. J. 2021; 131 (11)

  Abstract

  Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.

  View details for DOI 10.1172/JCI146256

  View details for PubMedID 34060485

• Eliminating hypoxic tumor cells improves response to PARP inhibitors in homologous recombination & ndash;deficient cancer models JOURNAL OF CLINICAL INVESTIGATION Mehibel, M., Xu, Y., Li, C. G., Moon, E., Thakkar, K. N., Diep, A. N., Kim, R. K., Bloomstein, J. D., Xiao, Y., Bacal, J., Saldivar, J. C., Le, Q., Cimprich, K. A., Rankin, E. B., Giaccia, A. J. 2021; 131 (11)

  View details for DOI 10.1172/JCI146256.

  View details for Web of Science ID 000656969100004

• -003 Oxygen dependent resistance to PARP inhibitors Mehibel, M., Xu, J., Li, G., Moon, J., Thakkar, K., Diep, A., Kim, R., Blomstein, J., Xiao, S., Bacal, J., Saldivar, J., Le, Q., Cimprich, K., Rankin, E., Giaccia, A. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000641160600004

• R-Loops as Cellular Regulators and Genomic Threats MOLECULAR CELL Crossley, M. P., Bocek, M., Cimprich, K. A. 2019; 73 (3): 398-411

  View details for DOI 10.1016/j.molcel.2019.01.024

  View details for Web of Science ID 000458015200004

• PPARgamma Interaction with UBR5/ATMIN Promotes DNA Repair to Maintain Endothelial Homeostasis. Cell reports Li, C. G., Mahon, C., Sweeney, N. M., Verschueren, E., Kantamani, V., Li, D., Hennigs, J. K., Marciano, D. P., Diebold, I., Abu-Halawa, O., Elliott, M., Sa, S., Guo, F., Wang, L., Cao, A., Guignabert, C., Sollier, J., Nickel, N. P., Kaschwich, M., Cimprich, K. A., Rabinovitch, M. 2019; 26 (5): 1333

  Abstract

  Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor gamma (PPARgamma) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARgamma promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARgamma depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARgamma DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARgamma signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARgamma-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARgamma DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH.

  View details for DOI 10.1016/j.celrep.2019.01.013

  View details for PubMedID 30699358

• An intrinsic S/G(2) checkpoint enforced by ATR SCIENCE Saldivar, J. C., Hamperl, S., Bocek, M. J., Chung, M., Bass, T. E., Cisneros-Soberanis, F., Samejima, K., Xie, L., Paulson, J. R., Earnshaw, W. C., Cortez, D., Meyer, T., Cimprich, K. A. 2018; 361 (6404): 806-809

  View details for DOI 10.1126/science.aap9346

  View details for Web of Science ID 000442828700047

• Faulty replication can sting NATURE Crossley, M. P., Cimprich, K. A. 2018; 557 (7703): 34–35

  View details for Web of Science ID 000431234500021

  View details for PubMedID 29713064

• A new mitotic activity comes into focus SCIENCE Saldivar, J. C., Cimprich, K. A. 2018; 359 (6371): 30–31

  View details for PubMedID 29302000

• Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage eLIFE Stork, C. T., Bocek, M., Crossley, M. P., Sollier, J., Sanz, L. A., Chédin, F., Swigut, T., Cimprich, K. A. 2016

  Abstract

  The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility.

  View details for DOI 10.7554/eLife.17548

  View details for PubMedCentralID PMC5030092

• DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome JOURNAL OF CLINICAL INVESTIGATION Slaats, G. G., Saldivar, J. C., Bacal, J., Zeman, M. K., Kile, A. C., Hynes, A. M., Srivastava, S., Nazmutdinova, J., den Ouden, K., Zagers, M. S., Foletto, V., Verhaar, M. C., Miles, C., Sayer, J. A., Cimprich, K. A., Giles, R. H. 2015; 125 (9): 3657-3666

  Abstract

  Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

  View details for DOI 10.1172/JCI80657

  View details for Web of Science ID 000362303600038

  View details for PubMedCentralID PMC4588305

• DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis. journal of cell biology Zeman, M. K., Lin, J., Freire, R., Cimprich, K. A. 2014; 206 (2): 183-197

  Abstract

  Deoxyribonucleic acid (DNA) lesions encountered during replication are often bypassed using DNA damage tolerance (DDT) pathways to avoid prolonged fork stalling and allow for completion of DNA replication. Rad18 is a central E3 ubiquitin ligase in DDT, which exists in a monoubiquitinated (Rad18•Ub) and nonubiquitinated form in human cells. We find that Rad18 is deubiquitinated when cells are treated with methyl methanesulfonate or hydrogen peroxide. The ubiquitinated form of Rad18 does not interact with SNF2 histone linker plant homeodomain RING helicase (SHPRH) or helicase-like transcription factor, two downstream E3 ligases needed to carry out error-free bypass of DNA lesions. Instead, it interacts preferentially with the zinc finger domain of another, nonubiquitinated Rad18 and may inhibit Rad18 function in trans. Ubiquitination also prevents Rad18 from localizing to sites of DNA damage, inducing proliferating cell nuclear antigen monoubiquitination, and suppressing mutagenesis. These data reveal a new role for monoubiquitination in controlling Rad18 function and suggest that damage-specific deubiquitination promotes a switch from Rad18•Ub-Rad18 complexes to the Rad18-SHPRH complexes necessary for error-free lesion bypass in cells.

  View details for DOI 10.1083/jcb.201311063

  View details for PubMedID 25023518

  View details for PubMedCentralID PMC4107794

• The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA repair Hamperl, S., Cimprich, K. A. 2014; 19: 84-94

  Abstract

  Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell.

  View details for DOI 10.1016/j.dnarep.2014.03.023

  View details for PubMedID 24746923

  View details for PubMedCentralID PMC4051866

• The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability DNA REPAIR Hamperl, S., Cimprich, K. A. 2014; 19: 84-94

  Abstract

  Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell.

  View details for DOI 10.1016/j.dnarep.2014.03.023

  View details for Web of Science ID 000338408200010

  View details for PubMedCentralID PMC4051866

• Whole-Exome Sequencing Reveals TopBP1 as a Novel Gene in Idiopathic Pulmonary Arterial Hypertension AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Perez, V. A., Yuan, K., Lyuksyutova, M. A., Dewey, F., Orcholski, M. E., Shuffle, E. M., Mathur, M., Yancy, L., Rojas, V., Li, C. G., Cao, A., Alastalo, T., Khazeni, N., Cimprich, K. A., Butte, A. J., Ashley, E., Zamanian, R. T. 2014; 189 (10): 1260-1272

  Abstract

  Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disorder characterized by progressive loss of pulmonary microvessels. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable and ∼15% of patients with IPAH, their low penetrance (∼20%) suggests that other unidentified genetic modifiers are required for manifestation of the disease phenotype. Use of whole-exome sequencing (WES) has recently led to the discovery of novel susceptibility genes in heritable PAH, but whether WES can also accelerate gene discovery in IPAH remains unknown.To determine whether WES can help identify novel gene modifiers in patients with IPAH.Exome capture and sequencing was performed on genomic DNA isolated from 12 unrelated patients with IPAH lacking BMPR2 mutations. Observed genetic variants were prioritized according to their pathogenic potential using ANNOVAR.A total of nine genes were identified as high-priority candidates. Our top hit was topoisomerase DNA binding II binding protein 1 (TopBP1), a gene involved in the response to DNA damage and replication stress. We found that TopBP1 expression was reduced in vascular lesions and pulmonary endothelial cells isolated from patients with IPAH. Although TopBP1 deficiency made endothelial cells susceptible to DNA damage and apoptosis in response to hydroxyurea, its restoration resulted in less DNA damage and improved cell survival.WES led to the discovery of TopBP1, a gene whose deficiency may increase susceptibility to small vessel loss in IPAH. We predict that use of WES will help identify gene modifiers that influence an individual's risk of developing IPAH.

  View details for DOI 10.1164/rccm.201310-17490C

  View details for Web of Science ID 000336017200018

  View details for PubMedID 24702692

  View details for PubMedCentralID PMC4225850

• Causes and consequences of replication stress. Nature cell biology Zeman, M. K., Cimprich, K. A. 2013; 16 (1): 2-9

  Abstract

  Replication stress is a complex phenomenon that has serious implications for genome stability, cell survival and human disease. Generation of aberrant replication fork structures containing single-stranded DNA activates the replication stress response, primarily mediated by the kinase ATR (ATM- and Rad3-related). Along with its downstream effectors, ATR stabilizes and helps to restart stalled replication forks, avoiding the generation of DNA damage and genome instability. Understanding this response may be key to diagnosing and treating human diseases caused by defective responses to replication stress.

  View details for DOI 10.1038/ncb2897

  View details for PubMedID 24366029

• PHD domain from human SHPRH JOURNAL OF BIOMOLECULAR NMR Machado, L. E., Pustovalova, Y., Kile, A. C., Pozhidaeva, A., Cimprich, K. A., Almeida, F. C., Bezsonova, I., Korzhnev, D. M. 2013; 56 (4): 393-399

  View details for DOI 10.1007/s10858-013-9758-2

  View details for Web of Science ID 000323661800011

  View details for PubMedID 23907177

• Finally, Polyubiquitinated PCNA Gets Recognized MOLECULAR CELL Zeman, M. K., Cimprich, K. A. 2012; 47 (3): 333-334

  Abstract

  Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, together with Weston et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA and plays an important role in restarting stalled replication forks.

  View details for DOI 10.1016/j.molcel.2012.07.024

  View details for Web of Science ID 000307484600002

  View details for PubMedID 22883622

• A Two-Dimensional ERK-AKT Signaling Code for an NGF-Triggered Cell-Fate Decision MOLECULAR CELL Chen, J., Lin, J., Cimprich, K. A., Meyer, T. 2012; 45 (2): 196-209

  Abstract

  Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.

  View details for DOI 10.1016/j.molcel.2011.11.023

  View details for PubMedID 22206868

• Checkpoint recovery after DNA damage: a rolling stop for CDKs EMBO REPORTS Duursma, A. M., Cimprich, K. A. 2010; 11 (6): 411-412

  View details for DOI 10.1038/embor.2010.76

  View details for Web of Science ID 000278234800005

  View details for PubMedID 20467435

  View details for PubMedCentralID PMC2892317

• HARPing on about the DNA damage response during replication GENES & DEVELOPMENT Driscoll, R., Cimprich, K. A. 2009; 23 (20): 2359-2365

  Abstract

  In this issue of Genes & Development, four papers report that the annealing helicase HepA-related protein (HARP, also known as SMARCAL1 [SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1]) binds directly to the ssDNA-binding protein Replication protein A (RPA) and is recruited to sites of replicative stress. Knockdown of HARP results in hypersensitivity to multiple DNA-damaging agents and defects in fork stability or restart. These exciting insights reveal a key new player in the S-phase DNA damage response.

  View details for DOI 10.1101/gad.1860609

  View details for Web of Science ID 000270849700001

  View details for PubMedID 19833762

  View details for PubMedCentralID PMC2764495

• Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA JOURNAL OF BIOLOGICAL CHEMISTRY Kochaniak, A. B., Habuchi, S., Loparo, J. J., Chang, D. J., Cimprich, K. A., Walter, J. C., van Oijen, A. M. 2009; 284 (26): 17700-17710

  Abstract

  Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.

  View details for DOI 10.1074/jbc.M109.008706

  View details for Web of Science ID 000267202500036

  View details for PubMedID 19411704

  View details for PubMedCentralID PMC2719409

• DNA damage tolerance: when it's OK to make mistakes NATURE CHEMICAL BIOLOGY Chang, D. J., Cimprich, K. A. 2009; 5 (2): 82-90

  Abstract

  Mutations can be beneficial under conditions in which genetic diversity is advantageous, such as somatic hypermutation and antibody generation, but they can also be lethal when they disrupt basic cellular processes or cause uncontrolled proliferation and cancer. Mutations arise from inaccurate processing of lesions generated by endogenous and exogenous DNA damaging agents, and the genome is particularly vulnerable to such damage during S phase. In this phase of the cell cycle, many lesions in the DNA template block replication. Such lesions must be bypassed in order to preserve fork stability and to ensure completion of DNA replication. Lesion bypass is carried out by a set of error-prone and error-free processes collectively referred to as DNA damage tolerance mechanisms. Here, we discuss how two types of DNA damage tolerance, translesion synthesis and template switching, are regulated at stalled replication forks by ubiquitination of PCNA, and the conditions under which they occur.

  View details for DOI 10.1038/nchembio.139

  View details for PubMedID 19148176

• ATR: an essential regulator of genome integrity NATURE REVIEWS MOLECULAR CELL BIOLOGY Cimprich, K. A., Cortez, D. 2008; 9 (8): 616-627

  Abstract

  Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.

  View details for DOI 10.1038/nrm2450

  View details for Web of Science ID 000257882000014

  View details for PubMedID 18594563

  View details for PubMedCentralID PMC2663384

• Probing ATR activation with model DNA templates CELL CYCLE Cimprich, K. A. 2007; 6 (19): 2348-2354

  Abstract

  The ATR kinase is a critical upstream component of a checkpoint pathway that responds to many forms of damaged and incompletely replicated DNA. Cellular processes such as DNA replication and repair are thought to convert these DNA lesions into a common DNA intermediate that activates this signaling pathway. Indeed, numerous studies have shown that two DNA structures formed during these processes--single-stranded DNA (ssDNA) and junctions between double-stranded DNA (dsDNA) and ssDNA--are important components of the ATR-activating structure. However, an unanswered question is whether primed ssDNA is sufficient for activation of the ATR response. We recently demonstrated that primed ssDNA is sufficient to induce a bona fide checkpoint response in Xenopus egg extracts. This is the first well-defined DNA structure capable of eliciting ATR activation. Using this structure, we examined the contribution of ds/ssDNA junctions and ssDNA to checkpoint activation. Our results indicate the context in which the checkpoint-activating structure is generated may contribute significantly to its signaling properties. Here we discuss the implications of our findings, in the context of other recent work in the field, on our understanding of checkpoint signaling.

  View details for Web of Science ID 000251085700008

  View details for PubMedID 17700074

• The ATR pathway: Fine-tuning the fork DNA REPAIR Paulsen, R. D., Cimprich, K. A. 2007; 6 (7): 953-966

  Abstract

  The proper detection and repair of DNA damage is essential to the maintenance of genomic stability. The genome is particularly vulnerable during DNA replication, when endogenous and exogenous events can hinder replication fork progression. Stalled replication forks can fold into deleterious conformations and are also unstable structures that are prone to collapse or break. These events can lead to inappropriate processing of the DNA, ultimately resulting in genomic instability, chromosomal alterations and cancer. To cope with stalled replication forks, the cell relies on the replication checkpoint to block cell cycle progression, downregulate origin firing, stabilize the fork itself, and restart replication. The ATR (ATM and Rad3-related) kinase and its downstream effector kinase, Chk1, are central regulators of the replication checkpoint. Loss of these checkpoint proteins causes replication fork collapse and chromosomal rearrangements which may ultimately predispose affected individuals to cancer. This review summarizes our current understanding of how the ATR pathway recognizes and stabilizes stalled replication forks.

  View details for DOI 10.1016/j.dnarep.2007.02.015

  View details for Web of Science ID 000248092000009

  View details for PubMedID 17531546

• Analyzing the ATR-mediated checkpoint using Xenopus egg extracts METHODS Lupardus, P. J., Van, C., Cimprich, K. A. 2007; 41 (2): 222-231

  Abstract

  Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.

  View details for DOI 10.1016/j.ymeth.2006.07.024

  View details for Web of Science ID 000243758100010

  View details for PubMedID 17189864

  View details for PubMedCentralID PMC2657337

• Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities JOURNAL OF BIOLOGICAL CHEMISTRY Chang, D. J., Lupardus, P. J., Cimprich, K. A. 2006; 281 (43): 32081-32088

  Abstract

  Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.

  View details for DOI 10.1074/jbc.M606799200

  View details for PubMedID 16959771

• Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling EMBO JOURNAL D Bomgarden, R., Lupardus, P. J., Soni, D. V., Yee, M., Ford, J. M., Cimprich, K. A. 2006; 25 (11): 2605-2614

  Abstract

  An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.

  View details for DOI 10.1038/sj.emboj.7601123

  View details for PubMedID 16675950

• Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of claspin and the Rad9 carboxy terminus MOLECULAR BIOLOGY OF THE CELL Lupardus, P. J., Cimprich, K. A. 2006; 17 (4): 1559-1569

  Abstract

  The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.

  View details for DOI 10.1091/mbc.E05-09-0865

  View details for Web of Science ID 000236657900007

  View details for PubMedID 16436514

  View details for PubMedCentralID PMC1415302

• Characterization of the minimal checkpoint-activating DNA structure Cimprich, K. A., Byun, T. S., MacDougall, C. FEDERATION AMER SOC EXP BIOL. 2006: A1360

  View details for Web of Science ID 000236326204487

• Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks MOLECULAR AND CELLULAR BIOLOGY Sobeck, A., Stone, S., Costanzo, V., De Graaf, B., Reuter, T., de Winter, J., Wallisch, M., Akkari, Y., Olson, S., Wang, W. D., Joenje, H., CHRISTIAN, J. L., Lupardus, P. J., Cimprich, K. A., Gautier, J., Hoatlin, M. E. 2006; 26 (2): 425-437

  Abstract

  Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

  View details for PubMedID 16382135

• A cell-permeable, activity-based probe for protein and lipid kinases JOURNAL OF BIOLOGICAL CHEMISTRY Yee, M., Fas, S. C., Stohlmeyer, M. M., Wandless, T. J., Cimprich, K. A. 2005; 280 (32): 29053-29059

  Abstract

  Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

  View details for DOI 10.1074/jbc.M504730200

  View details for Web of Science ID 000231021300030

  View details for PubMedID 15975929

• G2 damage checkpoints: what is the turn-on? JOURNAL OF CELL SCIENCE O'Connell, M. J., Cimprich, K. A. 2005; 118 (1): 1-6

  Abstract

  Cells mount a coordinated response to DNA damage, activating DNA repair pathways and cell-cycle checkpoint pathways to allow time for DNA repair to occur. In human cells, checkpoint responses can be divided into p53-dependent and p53-independent pathways, the latter being predominant in G2 phase of the cell cycle. The p53-independent pathway involves a phosphorylation cascade that activates the Chk1 effector kinase and induces G2 arrest through inhibitory tyrosine phosphorylation of Cdc2. At the top of this cascade are the ATR and ATM kinases. How ATM and ATR recognize DNA damage and activate this checkpoint pathway is only beginning to emerge. Single-stranded DNA, a result of stalled DNA replication or processing of chromosomal lesions, appears to be central to the activation of ATR. The recruitment of replication protein A to single-stranded DNA facilitates the recruitment of several complexes of checkpoint proteins. In this context, ATR is activated and then phosphorylates the C-terminus of Chk1, activating it to enforce a block to mitotic entry.

  View details for DOI 10.1242/jcs.01626

  View details for Web of Science ID 000226931500001

  View details for PubMedID 15615778

• Replication-dependent activation of the DNA damage checkpoint. Byun, T. S., Yee, M. C., Cimprich, K. A. AMER CHEMICAL SOC. 2004: U180

  View details for Web of Science ID 000223712800749

• Checkpoint adaptation: Molecular mechanisms uncovered CELL Lupardus, P. J., Cimprich, K. A. 2004; 117 (5): 555-556

  Abstract

  Adaptation to the DNA damage checkpoint is a phenomenon long thought to be confined to the unicellular world. A new report in this issue of Cell by suggests the presence of a checkpoint adaptation pathway in Xenopus egg extracts that displays interesting molecular parallels to adaptation in yeast.

  View details for Web of Science ID 000221857900001

  View details for PubMedID 15163402

• Fragile sites: Breaking up over a slowdown CURRENT BIOLOGY Cimprich, K. A. 2003; 13 (6): R231-R233

  Abstract

  Common fragile sites are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Recent data suggest that these sites depend on the checkpoint kinase ATR to maintain their stability.

  View details for DOI 10.1016/S0960-9822(03)00158-1

  View details for Web of Science ID 000181696800011

  View details for PubMedID 12646149

• Enforced proximity in the function of a famous scaffold MOLECULAR CELL Ferrell, J. E., Cimprich, K. A. 2003; 11 (2): 289-291

  Abstract

  Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.

  View details for Web of Science ID 000181296800005

  View details for PubMedID 12620218

• An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication MOLECULAR CELL Costanzo, V., Shechter, D., Lupardus, P. J., Cimprich, K. A., Gottesman, M., Gautier, J. 2003; 11 (1): 203-213

  Abstract

  We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.

  View details for Web of Science ID 000180848900022

  View details for PubMedID 12535533

• Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Post, S., Weng, Y. C., Cimprich, K., Chen, L. B., Xu, Y., Lee, E. Y. 2001; 98 (23): 13102-13107

  Abstract

  ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM- and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser(635) and Ser(645) in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G(1), S, and G(2)/M, but not in early-mid G(1). Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G(1). Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser(645) was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G(1)/S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells.

  View details for Web of Science ID 000172076800050

  View details for PubMedID 11687627

  View details for PubMedCentralID PMC60831

• Xenopus ATR is a replication-dependent chromatin-binding protein required for the DMA replication checkpoint CURRENT BIOLOGY Hekmat-Nejad, M., You, Z. S., Yee, M. C., Newport, J. W., Cimprich, K. A. 2000; 10 (24): 1565-1573

  Abstract

  The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available.We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation.ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.

  View details for Web of Science ID 000166807100016

  View details for PubMedID 11137007

• Activation of the ATM kinase by ionizing radiation and phosphorylation of p53 SCIENCE Canman, C. E., Lim, D. S., Cimprich, K. A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M. B., Siliciano, J. D. 1998; 281 (5383): 1677-1679

  Abstract

  The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

  View details for Web of Science ID 000075856500048

  View details for PubMedID 9733515

• Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints EMBO JOURNAL Cliby, W. A., Roberts, C. J., Cimprich, K. A., Stringer, C. M., LAMB, J. R., Schreiber, S. L., Friend, S. H. 1998; 17 (1): 159-169

  Abstract

  ATR, a phosphatidylinositol kinase-related protein homologous to ataxia telangiectasia mutated (ATM), is important for the survival of human cells following many forms of DNA damage. Expression of a kinase-inactive allele of ATR (ATRkd) in human fibroblasts causes increased sensitivity to ionizing radiation (IR), cis-platinum and methyl methanesulfonate, but only slight UV radiation sensitivity. ATRkd overexpression abrogates the G2/M arrest after exposure to IR, and overexpression of wild-type ATR complements the radioresistant DNA synthesis phenotype of cells lacking ATM, suggesting a potential functional overlap between these proteins. ATRkd overexpression also causes increased sensitivity to hydroxyurea that is associated with microtubule-mediated nuclear abnormalities. These observations are consistent with uncoupling of certain mitotic events from the completion of S-phase. Thus, ATR is an important component of multiple DNA damage response pathways and may be involved in the DNA replication (S/M) checkpoint.

  View details for Web of Science ID 000071708000015

  View details for PubMedID 9427750

• cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cimprich, K. A., Shin, T. B., Keith, C. T., Schreiber, S. L. 1996; 93 (7): 2850-2855

  Abstract

  A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

  View details for Web of Science ID A1996UD37500043

  View details for PubMedID 8610130