Honors & Awards
Ellison Senior Scholar Award, Ellison Foundation (2009)
Leukemia and Lymphoma Scholar Award, Leukemia and Lymphoma Society (2004)
Beckman Scholar Award, Arnold and Mabel Beckman Foundation (2000)
Burroughs Wellcome New Investigator Award in Toxicology, Burroughs Wellcome Foundation (1999)
Kimmel Scholar Award, Kimmel Foundation (1998)
B.S., University of Notre Dame, Chemistry (1989)
Ph.D., Harvard University, Chemistry (1994)
Current Research and Scholarly Interests
Genomic instability contributes to many diseases, such as cancer, neurodegenerative disease, and developmental disorders, but it also underlies many natural processes including aging, evolution, and antibody diversification. The Cimprich lab is focused on understanding how cells maintain genomic stability, with an emphasis on the DNA damage response (DDR). This is a complex, multi-faceted response that requires cells to sense the presence of DNA damage within the genome, as well as to “choose” and coordinate a range of downstream events and outcomes. These include effects on DNA repair, transcription, and DNA replication, as well as cell cycle arrest, apoptosis, and senescence.
We are particularly interested in understanding how DNA damage is identified and resolved during DNA replication, when the genome is particularly vulnerable due to stalling of the replication fork at naturally arising and induced DNA lesions, structures or protein-DNA complexes. Stalled replication forks are unstable and can be processed in aberrant ways, leading to double-strand break formation, which is known to drive chromosomal translocation and rearrangements in cancer cells. Indeed, replication stress, a result of slowing replication fork progression, is commonly observed in cancer cells due to loss of the DDR or oncogene activation.
The lab studies the DDR using cultured mammalian cells, as well as cell-free extracts derived from the eggs of the frog Xenopus laevis together with a variety of techniques. Our goal is to understand how the DDR is initiated, how this pathway is integrated with the processes of DNA replication and transcription, and how cells recover from DNA damage.
Specific areas of current interest are:
Checkpoint Activation and Signaling. We are interested in understanding how the DDR is initiated, and how it regulates DNA replication and DNA repair. In particular, we are interested in how stalled replication forks are stabilized, so as to prevent further DNA damage, and how replication resumes when forks stall frequently as in cancer cells. We have identified new factors involved in these processes and ongoing projects relate to studying the roles of these factors and other known DDR proteins in these processes.
DNA Damage Tolerance. DNA damage tolerance (DDT) pathways promote the completion of replication when a fork stalls by allowing bypass of the lesion, an event that leaves repair to a more convenient time. Although DDT pathways suppress fork collapse, thereby avoiding deleterious DNA breaks, bypass can be mutagenic. We are interested in understanding the mechanisms involved in DDT, and how the cell balances mutagenic and non-mutagenic DDT pathways.
New Pathways for Genome Stability/Human Disease: Recently, we performed a genome-wide siRNA screen to define the processes and proteins that protect cells from DNA damage, particularly during DNA replication. We are currently characterizing proteins identified in this screen with interesting ties to genome maintenance and human disease. One such protein is Nek8/NPHP9, a protein kinase we have shown to prevent DNA damage at stalled forks by regulating CDK activity. Our findings have provided exciting molecular insight into a novel link between the replication stress response and kidney diseases, including renal ciliopathies.
Transcription, RNA, and DNA Damage. Results from our genome-wide screen indicate that RNA is a prominent and underexplored source of genome instability in cell. Specifically, we found that defects in some RNA processing genes lead to DNA damage through the formation of toxic RNA-DNA hybrids and R-loops. We are interested in studying how these structures arise, how they lead to DNA damage, and their physiological function. We are also interested in understanding how cells deal with transcription complexes during DNA replication, as these can act as replication fork barriers and promote fork arrest.
Independent Studies (13)
- Directed Instruction/Reading
CHEM 110 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Introduction to Methods of Investigation
CHEM 190 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr, Sum)
- Out-of-Department Directed Reading
BIO 198X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr, Sum)
- Research and Special Advanced Work
CHEM 200 (Aut, Win, Spr, Sum)
- Research in Chemistry
CHEM 301 (Aut, Win, Spr, Sum)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Directed Instruction/Reading
- Prior Year Courses
NEK8 Links the ATR-Regulated Replication Stress Response and S Phase CDK Activity to Renal Ciliopathies.
2013; 51 (4): 423-439
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
2013; 27 (14): 1610-1623
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
A Role for the MRN Complex in ATR Activation via TOPBP1 Recruitment
2013; 50 (1): 116-122
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
SHPRH and HLTF Act in a Damage-Specific Manner to Coordinate Different Forms of Postreplication Repair and Prevent Mutagenesis
2011; 42 (2): 237-249
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 ?, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase ?. 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
Continued primer synthesis at stalled replication forks contributes to checkpoint activation
JOURNAL OF CELL BIOLOGY
2010; 189 (2): 233-246
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 Web of Science ID 000276825200008
View details for PubMedID 20385778
A Genome-wide siRNA Screen Reveals Diverse Cellular Processes and Pathways that Mediate Genome Stability
2009; 35 (2): 228-239
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
The structural determinants of checkpoint activation
GENES & DEVELOPMENT
2007; 21 (8): 898-903
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
Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint
GENES & DEVELOPMENT
2005; 19 (9): 1040-1052
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
A novel protein activity mediates DNA binding of an ATR-ATRIP complex
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (14): 13346-13353
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
2003; 13 (12): 1047-1051
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
2002; 16 (18): 2327-2332
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
Finally, Polyubiquitinated PCNA Gets Recognized
2012; 47 (3): 333-334
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
2012; 45 (2): 196-209
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 Web of Science ID 000299761700008
View details for PubMedID 22206868
- Checkpoint recovery after DNA damage: a rolling stop for CDKs EMBO REPORTS 2010; 11 (6): 411-412
HARPing on about the DNA damage response during replication
GENES & DEVELOPMENT
2009; 23 (20): 2359-2365
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
Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA
JOURNAL OF BIOLOGICAL CHEMISTRY
2009; 284 (26): 17700-17710
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
DNA damage tolerance: when it's OK to make mistakes
NATURE CHEMICAL BIOLOGY
2009; 5 (2): 82-90
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 Web of Science ID 000263090700010
View details for PubMedID 19148176
ATR: an essential regulator of genome integrity
NATURE REVIEWS MOLECULAR CELL BIOLOGY
2008; 9 (8): 616-627
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
Probing ATR activation with model DNA templates
2007; 6 (19): 2348-2354
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
2007; 6 (7): 953-966
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
2007; 41 (2): 222-231
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
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
2006; 281 (43): 32081-32088
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 Web of Science ID 000241414500005
View details for PubMedID 16959771
Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling
2006; 25 (11): 2605-2614
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 Web of Science ID 000238709400032
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
2006; 17 (4): 1559-1569
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
Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (2): 425-437
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 Web of Science ID 000234676100004
View details for PubMedID 16382135
A cell-permeable, activity-based probe for protein and lipid kinases
JOURNAL OF BIOLOGICAL CHEMISTRY
2005; 280 (32): 29053-29059
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
2005; 118 (1): 1-6
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
Checkpoint adaptation: Molecular mechanisms uncovered
2004; 117 (5): 555-556
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
2003; 13 (6): R231-R233
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
2003; 11 (2): 289-291
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
2003; 11 (1): 203-213
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
2001; 98 (23): 13102-13107
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
Xenopus ATR is a replication-dependent chromatin-binding protein required for the DMA replication checkpoint
2000; 10 (24): 1565-1573
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
1998; 281 (5383): 1677-1679
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
1998; 17 (1): 159-169
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
1996; 93 (7): 2850-2855
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