Biology subjects

Symington, L. S.

Publications and source records attributed to Symington, L. S..

8 recordsLinked to original sources

bioRxiv2025

Asymmetrical recognition and processing of double-strand breaks formed during DNA replication

DNA end resection to generate 3' ssDNA overhangs is the first step in homology-directed mechanisms of double-strand break (DSB) repair. While end resection has been extensively studied in the repair of endonuclease-induced DSBs, little is known about how resection proceeds at DSBs generated during DNA replication. We previously established a system to generate replication-dependent double-ended DSBs at the sites of nicks induced by the Cas9D10A nickase in the budding yeast genome. Here, we suggest that these DSBs form in an asymmetric manner, with one break end being blunt or near blunt, and the other bearing a 3' ssDNA overhang of up the size of an Okazaki fragment. We find that Mre11 preferentially binds blunt ends and is required for the removal Ku from these DSB ends. In contrast, the ends predicted to have 3' overhangs have minimal Ku binding, and end resection at these break ends can proceed in a mostly Mre11-independent manner through either the Exo1 or Dna2-Sgs1 long-range resection pathways. These findings indicate that resection proceeds differently at replication-dependent DSBs than at canonical DSBs, and reveals that Ku selectively binds nearly blunt ends, potentially explaining why replication-dependent DSBs are poorly repaired by non-homologous end joining. Significance StatementDNA end resection is a critical early step in the repair of DNA double-strand breaks by homologous recombination. Studies using endonucleases that generate DSBs in all phases of the cell cycle have shown that end resection proceeds by the sequential action of the Mre11 complex and long-range resection nucleases, Exo1 or Dna2-Sgs1. In this study, we restricted DSB generation to S-phase and found that the genetic requirements for end resection at more physiologically relevant DSBs are different to those at endonuclease-induced DSBs. The asymmetrical recognition and processing of replication-dependent DSBs may also explain the bias toward homologous recombination mediated repair over non-homologous end joining.

genetics↗

bioRxiv2024

Strand asymmetry in the repair of replication dependent double-strand breaks

Single-strand breaks (SSBs) are one of the most common endogenous lesions and have the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate the mechanism of replication fork collapse at SSBs and subsequent repair, we employed Cas9 nickase (nCas9) to generate site and strand-specific nicks in the budding yeast genome. We show that nCas9-induced nicks are converted to mostly double-ended DSBs during S-phase. We find that repair of replication-dependent DSBs requires homologous recombination (HR) and is independent of canonical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister chromatid template, we observe minimal induction of inter-chromosomal HR by nCas9. Using nCas9 and a gRNA to nick either the leading or lagging strand template, we carried out a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs. All the core HR genes were recovered in the screen with both gRNAs, but we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway with only the gRNA targeting the leading strand template. By use of additional gRNAs, we find that the RCNA pathway is especially important to repair a leading strand fork collapse.

genetics↗

bioRxiv2023

Cohesin still drives homologous recombination repair of DNA double-strand breaks in late mitosis.

The cohesin complex maintains sister chromatid cohesion from S phase to anaphase onset. Cohesin also plays roles in chromosome structure and DNA repair. At anaphase onset, the cohesin subunit Scc1 is cleaved to allow segregation in an orderly manner, although some residual cohesin subunits remain to maintain chromosome structure. Efficient DNA double strand break (DSB) repair by homologous recombination (HR) with the sister chromatid also depends on cohesin. Here, we have examined the role of residual cohesin in DSB repair in telophase (late mitosis). We have found that Scc1 returns in telophase after DSBs, and that it partially reconstitutes a chromatin-bound cohesin complex with Smc1 and an acetylated pool of Smc3 after a single HO-induced DSB at the MAT locus. However, this new cohesin is neither required for the HR-driven MAT switching nor binds to the MAT locus after the DSB.

molecular biology↗

bioRxiv2023

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase delta and Rad51-dependent mechanism

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease. Foldback priming at naturally occurring inverted repeats is one mechanism proposed for the generation of inverted duplications. However, the initiating lesion for these events and the mechanism by which they form has not been fully elucidated. Here, we show that a DNA double-strand break (DSB) induced near natural short, inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase {delta} proof-reading activity acts non-redundantly with Rad1 nuclease to remove heterologous tails formed during foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol {delta} catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. Stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences and requires Rad51.

genetics↗

bioRxiv2022

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation

Homologous recombination (HR), the error-free mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5'-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely-linked repeats but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae. This requirement for long-range resection is coupled to DNA damage checkpoint activation and the need for checkpoint proteins. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1{Delta} sgs1{Delta} cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1{Delta} sgs1{Delta} cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

genetics↗

bioRxiv2021

Homologous recombination induced by a replication fork barrier requires cooperation between strand invasion and strand annealing activities

Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. Elucidating the mechanism of recombination between repeated sequences in the context of replication stress is essential to understanding how genome rearrangements occur. To gain insight into this process, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Remarkably, we show that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, as well as Mph1/Rad5 fork remodelers, Mre11/Exo1 short and long-range resection machineries, Rad1-Rad10 nuclease and DNA polymerase {delta}. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 structure-selective nucleases. Physical analysis of replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats at stalled replication forks that can actively contribute to genomic rearrangements.

genetics↗

bioRxiv2018

Xrs2 and Tel1 independently contribute to MR-mediated DNA tethering and replisome stability

The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling and catalytic functions in the cellular response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50, and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1 and it fails to tether DSBs resulting in sensitivity to genotoxins, replisome instability and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering, stalled replication fork stability and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.\n\nHighlightsO_LIXrs2 is required for recruitment but not for activation of Tel1 kinase\nC_LIO_LITel1 and Xrs2 function independently to optimize MR activity at DSBs and stalled replication forks\nC_LIO_LIStable association of Mre11 at DSBs is required to maintain end-to-end tethering\nC_LIO_LIMR-mediated DNA tethering promotes replisome stability and genome integrity\nC_LI

genetics↗

bioRxiv2018

Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5-3 resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5 strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2{Delta} mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2{Delta} cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2{Delta} is synthetic lethal with sgs1{Delta}, whereas the mre11-nd sgs1{Delta} mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1 mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2{Delta} mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

genetics↗