Alexandra (Sasha) Khristich

All Publications

• Replication-independent instability of Friedreich's ataxia GAA repeats during chronological aging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Neil, A. J., Hisey, J. A., Quasem, I., McGinty, R. J., Hitczenko, M., Khristich, A. N., Mirkin, S. M. 2021; 118 (5)

  Abstract

  Nearly 50 hereditary diseases result from the inheritance of abnormally long repetitive DNA microsatellites. While it was originally believed that the size of inherited repeats is the key factor in disease development, it has become clear that somatic instability of these repeats throughout an individual's lifetime strongly contributes to disease onset and progression. Importantly, somatic instability is commonly observed in terminally differentiated, postmitotic cells, such as neurons. To unravel the mechanisms of repeat instability in nondividing cells, we created an experimental system to analyze the mutability of Friedreich's ataxia (GAA)n repeats during chronological aging of quiescent Saccharomyces cerevisiae Unexpectedly, we found that the predominant repeat-mediated mutation in nondividing cells is large-scale deletions encompassing parts, or the entirety, of the repeat and adjacent regions. These deletions are caused by breakage at the repeat mediated by mismatch repair (MMR) complexes MutSβ and MutLα and DNA endonuclease Rad1, followed by end-resection by Exo1 and repair of the resulting double-strand breaks (DSBs) via nonhomologous end joining. We also observed repeat-mediated gene conversions as a result of DSB repair via ectopic homologous recombination during chronological aging. Repeat expansions accrue during chronological aging as well-particularly in the absence of MMR-induced DSBs. These expansions depend on the processivity of DNA polymerase δ while being counteracted by Exo1 and MutSβ, implicating nick repair. Altogether, these findings show that the mechanisms and types of (GAA)n repeat instability differ dramatically between dividing and nondividing cells, suggesting that distinct repeat-mediated mutations in terminally differentiated somatic cells might influence Friedreich's ataxia pathogenesis.

  View details for DOI 10.1073/pnas.2013080118

  View details for Web of Science ID 000615345200019

  View details for PubMedID 33495349

  View details for PubMedCentralID PMC7865128

• On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability JOURNAL OF BIOLOGICAL CHEMISTRY Khristich, A. N., Mirkin, S. M. 2020; 295 (13): 4134-4170

  View details for DOI 10.1074/jbc.REV119.007678

  View details for Web of Science ID 000523442500007

• On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability. The Journal of biological chemistry Khristich, A. N., Mirkin, S. M. 2020; 295 (13): 4134-4170

  Abstract

  Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?

  View details for DOI 10.1074/jbc.REV119.007678

  View details for PubMedID 32060097

  View details for PubMedCentralID PMC7105313

• Large-scale contractions of Friedreich's ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Khristich, A. N., Armenia, J. F., Matera, R. M., Kolchinski, A. A., Mirkin, S. M. 2020; 117 (3): 1628-1637

  Abstract

  Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175-2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat's triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.

  View details for DOI 10.1073/pnas.1913416117

  View details for Web of Science ID 000508977600057

  View details for PubMedID 31911468

  View details for PubMedCentralID PMC6983365

• RNA-DNA hybrids promote the expansion of Friedreich's ataxia (GAA)(n) repeats via break-induced replication NUCLEIC ACIDS RESEARCH Neil, A. J., Liang, M. U., Khristich, A. N., Shah, K. A., Mirkin, S. M. 2018; 46 (7): 3487-3497

  Abstract

  Expansion of simple DNA repeats is responsible for numerous hereditary diseases in humans. The role of DNA replication, repair and transcription in the expansion process has been well documented. Here we analyzed, in a yeast experimental system, the role of RNA-DNA hybrids in genetic instability of long (GAA)n repeats, which cause Friedreich's ataxia. Knocking out both yeast RNase H enzymes, which counteract the formation of RNA-DNA hybrids, increased (GAA)n repeat expansion and contraction rates when the repetitive sequence was transcribed. Unexpectedly, we observed a similar increase in repeat instability in RNase H-deficient cells when we either changed the direction of transcription-replication collisions, or flipped the repeat sequence such that the (UUC)n run occurred in the transcript. The increase in repeat expansions in RNase H-deficient strains was dependent on Rad52 and Pol32 proteins, suggesting that break-induced replication (BIR) is responsible for this effect. We conclude that expansions of (GAA)n repeats are induced by the formation of RNA-DNA hybrids that trigger BIR. Since this stimulation is independent of which strand of the repeat (homopurine or homopyrimidine) is in the RNA transcript, we hypothesize that triplex H-DNA structures stabilized by an RNA-DNA hybrid (H-loops), rather than conventional R-loops, could be responsible.

  View details for DOI 10.1093/nar/gky099

  View details for Web of Science ID 000431137900023

  View details for PubMedID 29447396

  View details for PubMedCentralID PMC5909440

• Multi-targeted effects of G4-aptamers and their antiproliferative activity against cancer cells BIOCHIMIE Ogloblina, A. M., Khristich, A. N., Karpechenko, N. Y., Semina, S. E., Belitsky, G. A., Dolinnaya, N. G., Yakubovskaya, M. G. 2018; 145: 163-173

  Abstract

  We selected and investigated nine G-quadruplex (G4)-forming aptamers originally designed against different proteins involved in the regulation of cellular proliferation (STAT3, nucleolin, TOP1, SP1, VEGF, and SHP-2) and considered to be potential anticancer agents. We showed that under physiological conditions all the aptamers form stable G4s of different topology. G4 aptamers designed against STAT3, nucleolin and SP1 inhibit STAT3 transcriptional activity in human breast adenocarcinoma MCF-7 cells, and all the studied aptamers inhibit TOP1-mediated relaxation of supercoiled plasmid DNA. STAT3 inhibition by G4 aptamer designed against SP1 protein provides a new explanation for the SP1 and STAT3 crosstalk described recently. We found some correlation between G4-mediated inhibition of the DNA replication and TOP1 activity. Four G4 aptamers from our dataset that appeared to be the strongest TOP1 inhibitors most efficiently decreased de novo DNA synthesis, by up to 79-87%. Seven G4 aptamers demonstrated significantly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells than on immortalized mammary epithelial MCF-10A cells. Pleiotropic properties of G4 aptamers and their high specificity against cancer cells observed for the majority of the studied G4 aptamers allowed us to present them as promising candidates for multi-targeted cancer therapy.

  View details for DOI 10.1016/j.biochi.2017.11.020

  View details for Web of Science ID 000425282800021

  View details for PubMedID 29208488