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1.
bioRxiv ; 2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37662271

RESUMO

The mammalian RAD52 protein is a DNA repair factor that has both strand annealing and recombination mediator activities, yet is dispensable for cell viability. To characterize genetic contexts that reveal dependence on RAD52 to sustain cell viability (i.e., synthetic lethal relationships), we performed genome-wide CRISPR knock-out screens. Subsequent secondary screening found that depletion of ERCC6L in RAD52-deficient cells causes reduced viability and elevated genome instability, measured as accumulation of 53BP1 into nuclear foci. Furthermore, loss of RAD52 causes elevated levels of anaphase ultrafine bridges marked by ERCC6L, and conversely depletion of ERCC6L causes elevated RAD52 foci both in prometaphase and interphase cells. These effects were enhanced with combination treatments using hydroxyurea and the topoisomerase IIα inhibitor ICRF-193, and the timing of these treatments are consistent with defects in addressing such stress in mitosis. Thus, loss of RAD52 appears to cause an increased reliance on ERCC6L in mitosis, and vice versa. Consistent with this notion, combined depletion of ERCC6L and disrupting G2/M progression via CDK1 inhibition causes a marked loss of viability in RAD52-deficient cells. We suggest that RAD52 and ERCC6L play compensatory roles in protecting genome stability in mitosis.

2.
Nucleic Acids Res ; 50(12): 6870-6889, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35748867

RESUMO

Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.


Assuntos
DNA , DNA/genética
3.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34789574

RESUMO

Extrachromosomal circular DNA (eccDNA) originates from linear chromosomal DNA in various human tissues under physiological and disease conditions. The genomic origins of eccDNA have largely been investigated using in vitro-amplified DNA. However, in vitro amplification obscures quantitative information by skewing the total population stoichiometry. In addition, the analyses have focused on eccDNA stemming from single-copy genomic regions, leaving eccDNA from multicopy regions unexamined. To address these issues, we isolated eccDNA without in vitro amplification (naïve small circular DNA, nscDNA) and assessed the populations quantitatively by integrated genomic, molecular, and cytogenetic approaches. nscDNA of up to tens of kilobases were successfully enriched by our approach and were predominantly derived from multicopy genomic regions including segmental duplications (SDs). SDs, which account for 5% of the human genome and are hotspots for copy number variations, were significantly overrepresented in sperm nscDNA, with three times more sequencing reads derived from SDs than from the entire single-copy regions. SDs were also overrepresented in mouse sperm nscDNA, which we estimated to comprise 0.2% of nuclear DNA. Considering that eccDNA can be integrated into chromosomes, germline-derived nscDNA may be a mediator of genome diversity.


Assuntos
DNA Circular , Células Germinativas , Animais , Cromossomos , DNA , Variações do Número de Cópias de DNA , Genoma Humano , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Duplicações Segmentares Genômicas , Espermatozoides
4.
Nucleic Acids Res ; 49(15): 8714-8731, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379776

RESUMO

Microhomology-mediated break-induced replication (MMBIR) is a DNA repair pathway initiated by polymerase template switching at microhomology, which can produce templated insertions that initiate chromosomal rearrangements leading to neurological and metabolic diseases, and promote complex genomic rearrangements (CGRs) found in cancer. Yet, how often templated insertions accumulate from processes like MMBIR in genomes is poorly understood due to difficulty in directly identifying these events by whole genome sequencing (WGS). Here, by using our newly developed MMBSearch software, we directly detect such templated insertions (MMB-TIs) in human genomes and report substantial differences in frequency and complexity of MMB-TI events between normal and cancer cells. Through analysis of 71 cancer genomes from The Cancer Genome Atlas (TCGA), we observed that MMB-TIs readily accumulate de novo across several cancer types, with particularly high accumulation in some breast and lung cancers. By contrast, MMB-TIs appear only as germline variants in normal human fibroblast cells, and do not accumulate as de novo somatic mutations. Finally, we performed WGS on a lung adenocarcinoma patient case and confirmed MMB-TI-initiated chromosome fusions that disrupted potential tumor suppressors and induced chromothripsis-like CGRs. Based on our findings we propose that MMB-TIs represent a trigger for widespread genomic instability and tumor evolution.


Assuntos
Reparo do DNA , Neoplasias/genética , Adenocarcinoma de Pulmão/genética , Fibroblastos , Genes Supressores de Tumor , Genoma Humano , Instabilidade Genômica , Humanos , Neoplasias Pulmonares/genética , Mutagênese Insercional , Pele/citologia , Software
5.
Nature ; 590(7847): 655-659, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33473214

RESUMO

Break-induced replication (BIR) repairs one-ended double-strand breaks in DNA similar to those formed by replication collapse or telomere erosion, and it has been implicated in the initiation of genome instability in cancer and other human diseases1,2. Previous studies have defined the enzymes that are required for BIR1-5; however, understanding of initial and extended BIR synthesis, and of how the migrating D-loop proceeds through known replication roadblocks, has been precluded by technical limitations. Here we use a newly developed assay to show that BIR synthesis initiates soon after strand invasion and proceeds more slowly than S-phase replication. Without primase, leading strand synthesis is initiated efficiently, but is unable to proceed beyond 30 kilobases, suggesting that primase is needed for stabilization of the nascent leading strand. DNA synthesis can initiate in the absence of Pif1 or Pol32, but does not proceed efficiently. Interstitial telomeric DNA disrupts and terminates BIR progression, and BIR initiation is suppressed by transcription proportionally to the transcription level. Collisions between BIR and transcription lead to mutagenesis and chromosome rearrangements at levels that exceed instabilities induced by transcription during normal replication. Together, these results provide fundamental insights into the mechanism of BIR and how BIR contributes to genome instability.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Saccharomyces cerevisiae , Cromossomos Fúngicos/genética , DNA Helicases/deficiência , DNA Primase/metabolismo , DNA Fúngico/biossíntese , DNA Polimerase Dirigida por DNA/deficiência , Instabilidade Genômica , Cinética , Mutagênese , Mutação , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Telômero/genética , Fatores de Tempo , Transcrição Gênica
6.
Methods Mol Biol ; 2153: 307-328, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840789

RESUMO

Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.


Assuntos
Cromossomos Fúngicos/genética , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Saccharomyces cerevisiae/fisiologia , Replicação do DNA , Mutação , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nucleic Acids Res ; 47(18): 9666-9684, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392335

RESUMO

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.


Assuntos
Cromossomos/genética , Citidina Desaminase/genética , Reparo do DNA/genética , Proteínas/genética , Recombinação Genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Humanos , Mutagênese/genética , Mutação , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
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