Microhomology Selection for Microhomology Mediated End Joining in Saccharomyces cerevisiae

Lee, Kihoon; Ji, Jeong‐Seon; Yoon, Kihoon; Che, Jun; Seol, Ja-Hwan; Lee, Sang Eun; Shim, Eun Yong

doi:10.3390/genes10040284

Cited by 11 publications

(7 citation statements)

References 39 publications

(66 reference statements)

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“…Previous research has suggested that DSB repair that involves DNA resection is highly mutagenic because ssDNA regions created during resection must be filled in once HR has completed [ 58 , 59 , 60 ]. Gap-filling, either by DNA polymerase δ or by translesion DNA polymerases such as Polζ have been shown to be responsible for mutation rates 1000-fold over background spontaneous mutation rates in canonical DSB repair [ 61 , 62 ].…”

Section: Resultsmentioning

confidence: 99%

A Rad51-independent pathway promotes single-strand template repair in gene editing

et al. 2020

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The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo . We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3’ side of the ssODN which can anneal directly to the resected DSB end compared to the 5’ end. In addition DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch very close to the 3’ end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

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Section: Resultsmentioning

confidence: 99%

A Rad51-independent pathway promotes single-strand template repair in gene editing

et al. 2020

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“…Similarly, in fission yeast, even a single heterologous base at the 3 end reduces MMEJ efficiency two-fold [17,87]. Microhomologies used in MMEJ in budding yeast are longer than in metazoans, requiring at least 8 complementary base pairs in one study [88]. In contrast, the microhomologies used in metazoan alt-EJ can be as short as 1 bp, likely due to the annealing action of polymerase theta.…”

Section: Microhomologymentioning

confidence: 99%

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

Hanscom

McVey

2020

Cells

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Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.

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“…Previous research has suggested that DSB repair that involves DNA resection is highly 169 mutagenic because ssDNA regions created during resection must be filled in once HR has 170 completed [50,51,52]. Gap-filling, either by DNA polymerase or by translesion DNA 171 polymerases such as Polz have been shown to be responsible for mutation rates 1000-fold over 172 background spontaneous mutation rates [53,54].…”

Section: Sstr Is Accompanied By Frequent Mutations In Adjacent Regionmentioning

confidence: 99%

A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing

Gallagher¹,

Pham²,

Tsai³

et al. 2020

Preprint

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1 2 The Rad51/RecA family of recombinases perform a critical function in typical repair of double-3 strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded 4 DNA (dsDNA) template sequence to facilitate repair. However, repair of a DSB using single 5 stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene 6 editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-7 independent events in Saccharomyces cerevisiae in two different assays. Gene editing events 8 were carried out either by creating a DSB with the site-specific HO endonuclease and repairing 9 the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron 10 system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We 11show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the 12 Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent 13 overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition 14 of Rad51 on Rad52's strand annealing activity; thus, deletion of RAD51 suppresses both the 15 srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another 16 DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp 17 dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in 18 which the ssODN carried several mismatches. In the absence of the mismatch repair protein, 19Msh2, we found that the fate of mismatches carried on the ssDNA template are very different 20 at the 3' end, which can anneal directly to the resected DSB end, compared to the 5' end. We 21 also find that DNA polymerase Pold's 3' to 5' proofreading activity frequently excises a 22 Rdh54/Tid1 assume important roles. Rad51-independent BIR also requires the Mre11-Rad50-51Xrs2 complex, whereas Rad51-mediated events and SSA are merely delayed by the absence of 52 these proteins [18]. A third Rad51-independent pathway, another form of BIR, operates to 53 maintain telomeres in the absence of telomerase (known as Type II events). Here too, Rad59 54 and the MRX complex, as well as Rad52, are necessary, whereas the Type I Rad51-dependent 55 telomere maintenance pathway does not require either Rad59 or MRX [22,23,24]. Similarly, 56 DSB repair by intramolecular gene conversion involving short (33-bp) regions of homology is 57 inhibited by Rad51 and is dependent on the MRX complex, Rad59, and Rdh54 [25]. Rad51-58 independent BIR pathways are also dependent on the Srs2 helicase that antagonizes loading of 59 Rad51 onto resected DSB ends [21,26]. 60Use of the RNA-guided CRISPR/Cas9 endonuclease has revolutionized gene editing in 61 eukaryotic systems ranging from yeast to mammals [27,28,29]. Guided endonucleases are 62 programmed to create site-specific DSBs that can be repaired by providing a homologous 63 template [30,31]. One approach that has been shown to be an e...

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Microhomology Selection for Microhomology Mediated End Joining in Saccharomyces cerevisiae

Cited by 11 publications

References 39 publications

A Rad51-independent pathway promotes single-strand template repair in gene editing

A Rad51-independent pathway promotes single-strand template repair in gene editing

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

A Rad51-Independent Pathway Promotes Single-Strand Template Repair in Gene Editing

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