The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system ...remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from
, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric
RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.
DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair ...of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.
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•Partial destabilization of RPA binding to ssDNA modestly decreases break repair•Stable RPA-ssDNA interaction is critical for break-induced replication and gap repair•The rfa1 BIR defect is suppressed by elimination of Sgs1-Dna2 catalyzed end resection•Rad51 overexpression suppresses the rfa1 BIR defect
RPA plays an important role during homology-dependent repair (HDR) to protect fragile single-stranded DNA (ssDNA) intermediates. Here, Ruff et al. show that partial destabilization of RPA binding to ssDNA has modest effects on efficient intra-chromosomal HDR reactions but causes profound defects in HDR events that involve long-lived ssDNA intermediates, such as break-induced replication (BIR). The BIR defect caused by dysfunctional RPA can be partially overcome by preventing excessive end resection or by Rad51 overexpression.
The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the 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 and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.
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•Xrs2 is required for recruitment but not for activation of Tel1 kinase•Tel1 and Xrs2 function independently to optimize MR activity•Stable association of Mre11 at DSBs is required to maintain end-to-end tethering•MR-mediated DNA tethering promotes replisome stability and genome integrity
Oh et al. show that Tel1 and Xrs2 function independently to optimize MR activity at double-strand breaks (DSBs) and stalled replication forks. Stable association of MR at DSBs maintains end-to-end tethering and correlates with DNA damage resistance, decreased replication stress, and suppression of genome rearrangements.
Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to ...conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.
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•Pol δ synthesizes both strands during BIR•Pol α-primase and DNA ligase I are required for BIR•DNA synthesis during BIR is independent of Pol ε•HydEn-seq technology is applied to DNA repair synthesis
Donnianni et al. elucidate DNA polymerase usage during break-induced replication (BIR). In contrast to conventional S phase DNA replication, in which DNA Pol ε synthesizes the leading strand and Pols α and δ synthesize the lagging strand, the authors show that Pol δ synthesizes both strands during BIR.
Replication protein A (RPA) is the main eukaryotic single‐stranded DNA (ssDNA) binding protein, having essential roles in all DNA metabolic reactions involving ssDNA. RPA binds ssDNA with high ...affinity, thereby preventing the formation of secondary structures and protecting ssDNA from the action of nucleases, and directly interacts with other DNA processing proteins. Here, we discuss recent results supporting the idea that one function of RPA is to prevent annealing between short repeats that can lead to chromosome rearrangements by microhomology‐mediated end joining or the formation of hairpin structures that are substrates for structure‐selective nucleases. We suggest that replication fork catastrophe caused by depletion of RPA could result from cleavage of secondary structures by nucleases, and that failure to cleave hairpin structures formed at DNA ends could lead to gene amplification. These studies highlight the important role RPA plays in maintaining genome integrity.
Intrachromosomal Recombination in Yeast Epshtein, Anastasiya; Symington, Lorraine S; Klein, Hannah L
Methods in molecular biology (Clifton, N.J.),
2021, Letnik:
2153
Journal Article
Spontaneous and induced mitotic recombinations are driven by lesions such as single-strand nicks and gaps and double-strand breaks in the genome. For regions of the genome that are not repetitive, ...spontaneous recombination rates are too low to be detected by simple screening and require reporters where a recombination product can be selected. This chapter describes commonly used types of reporters where a gene is duplicated as direct repeats and both copies are mutated with different mutations, rendering the cell defective for the gene and auxotrophic for the gene product. Recombination between the two defective copies can result in a wild-type gene and a prototrophic phenotype for the cell. Methods to use these types of reporters to determine recombination rates between the two gene copies are described, and their use in monitoring both increased and decreased recombinations is discussed.
Recognition for Discoveries in DNA Repair Klein, Hannah L; Symington, Lorraine S
The New England journal of medicine,
08/2019, Letnik:
381, Številka:
7
Journal Article
Recenzirano
The understanding of mechanisms of DNA repair has provided a foundation on which to develop new approaches to cancer treatment and genetic engineering. This year’s Shaw Prize, awarded to Dr. Maria ...Jasin, recognizes the seminal nature of her discoveries for these fields.
Significance Chromosomal double-strand breaks (DSBs) are cytotoxic forms of DNA damage that must be accurately repaired to maintain genome integrity. The conserved Mre11–Rad50–Xrs2/NBS1 ...nuclease/ATPase complex plays an important role in repair by functioning as a damage sensor and by regulation of DNA end processing to ensure repair by the most appropriate mechanism. Yeast Sae2 is known to function with Mre11 to process DNA ends, but its precise role is poorly understood. Here we show that it is the failure to remove Mre11 from DNA ends, leading to persistent DNA damage signaling and cell cycle arrest, that causes sensitivity of Sae2-deficient cells to DNA damaging agents.
The Mre11–Rad50–Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2 Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2 Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M ᴾ¹¹⁰ᴸRX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2 Δ cells to clastogens.
Making the cut Symington, Lorraine S.
Nature (London),
10/2014, Letnik:
514, Številka:
7520
Journal Article
Recenzirano
Because MRX clipping is dependent on ATP, and a specific point mutation in the RAD50 gene, like loss of Sae2, prevents DNA clipping13, it is likely that Sae2 acts through Rad50 to activate Mre11.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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