Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

Lee, Miju; Lee, Chul-Hwan; Demin, Annie Albert; Munashingha, Palinda Ruvan; Amangyeld, Tamir; Kwon, Buki; Formosa, Tim; Seo, Yeon‐Soo

doi:10.1074/jbc.m114.548388

Cited by 19 publications

(19 citation statements)

References 105 publications

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“…Since MMS treatment leads to replication stress and most known suppressors of srs2 Δ reduce Rad51 nucleoprotein filament formation [78,91,92], we propose that SUMOylation of Rad59 is important for restraining toxic Rad51-mediated recombination during replication stress. This conclusion goes well with other reports of a role for Rad59 in DNA replication stress tolerance [3,93,94]. Similarly, the fact that we observed reduced recombination and increased MMS and Zeocin survival of srs2 Δ cells when mimicking constitutive SUMOylation of Rad52 is in agreement with the recent finding that toxic Rad51-dependent HR structures fail to form in srs2Δ rad52-SMT3ΔGG [78] and with the reduced recruitment of Rad51 to foci in the rad52-SMT3ΔGG rad59-SMT3ΔGG mutant (Fig.…”

Section: Discussionsupporting

confidence: 93%

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

et al. 2016

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Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.

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Section: Discussionsupporting

confidence: 93%

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

et al. 2016

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“…Rad51-independent HDR has also been reported for CRISPR-Cas9 (Collonnier et al, 2017). Rad52 and Rad59 are involved in Rad51-independent processing of Okazaki fragments (Lee et al, 2014), and the loss of detectable ARF in rad59 Δ and rad52 Δ rad59 Δ compared to the neutral effect of its paralog ( rad52 Δ) suggests a unique role for Rad59 to promote annealing of ssODNs at the replication fork. Although Rad52 may contribute to ssODN annealing at the replication fork, it also mediates loading of Rad51 to RPA-bound ssDNA (Song and Sung, 2000) whereas Rad59 does not contain a known Rad51 binding domain (Erler et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Barbieri

Muir

Akhuetie-Oni

et al. 2017

Cell

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SUMMARY We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing of synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with efficiencies >40% without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous β-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

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“…Both mutants are unable to suppress pol30-K164R (Figure 3C), implying that the suppression effect acts through the Rad52 DNA annealing ability. The rad52-Δ327 allele (45) lacks the C-ter of Rad52, which is required for interaction with Rad51. The rad52-QDDD-AAAA allele (45) is impaired in its interaction with RPA and recombination mediator activity.…”

Section: Resultsmentioning

confidence: 99%

“…The rad52-Δ327 allele (45) lacks the C-ter of Rad52, which is required for interaction with Rad51. The rad52-QDDD-AAAA allele (45) is impaired in its interaction with RPA and recombination mediator activity. Both alleles are thus unable to stimulate Rad51 activities.…”

Section: Resultsmentioning

confidence: 99%

Access to PCNA by Srs2 and Elg1 controls the choice between alternative repair pathways in yeast

Arbel

Bronstein

et al. 2020

Preprint

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During DNA replication stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here we show that in addition to trans-lesion synthesis and strand-transfer DTT mechanisms, an alternative repair mechanism (“salvage recombination”) that copies information from the sister chromatid, is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.

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Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

Cited by 19 publications

References 105 publications

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Access to PCNA by Srs2 and Elg1 controls the choice between alternative repair pathways in yeast

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