scholarly journals Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: different requirements for the RAD1, RAD10, and RAD52 genes.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 109-123 ◽  
Author(s):  
F Prado ◽  
A Aguilera
Keyword(s):  
Direct Repeat ◽  
Repeat Sequence ◽  
Single Strand ◽  
Dna Repeats ◽  
Recombination Mechanism ◽  
Reciprocal Exchange ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Strand Annealing

Abstract We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB)-induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.

Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated

1992 ◽  
Vol 12 (3) ◽  
pp. 1292-1303
Author(s):  
J Fishman-Lobell ◽  
N Rudin ◽  
J E Haber
Keyword(s):  
Direct Repeat ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Strand Breaks ◽  
Alternative Pathways ◽  
Deletion Formation ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Competing Pathways

HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells.

scholarly journals Effects of DNA Structure and Homology Length on Vaccinia Virus Recombination

2001 ◽  
Vol 75 (15) ◽  
pp. 6923-6932 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Dna Sequences ◽  
Accurate Method ◽  
Single Strand ◽  
Recombination Mechanism ◽  
Virus Recombination ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting ∼4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3′-to-5′ exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.

scholarly journals An Allele of RFA1 Suppresses RAD52-Dependent Double-Strand Break Repair in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 447-458 ◽  
Author(s):  
Julianne Smith ◽  
Rodney Rothstein
Keyword(s):  
Direct Repeat ◽  
Strand Break ◽  
Product Formation ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Recombination Mechanism ◽  
Formation Kinetics ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Sister Chromatid Recombination

Abstract An allele of RFA1, the largest subunit of the single-stranded DNA-binding complex RP-A, was identified as a suppressor of decreased direct-repeat recombination in rad1 rad52 double mutants. In this study, we used two LEU2 direct-repeat assays to investigate the mechanism by which the rfa1-D228Y allele increases recombination. We found that both intrachromatid and sister chromatid recombination are stimulated in rfa1-D228Y strains. In a rad1 rad52 background, however, the majority of the increased recombination is caused by stimulation of deletion events by an intrachromatid recombination mechanism that is likely to be single-strand annealing. Studies in which an HO endonuclease cut was introduced between the two leu2 copies indicate that the rfa1-D228Y mutation partially suppresses the rad52 defect in recovering recombination products. Furthermore, molecular analysis of processing and product formation kinetics reveals that, in a rad52 background, the rfa1-D228Y mutation results in increased levels of recombinant products and the disappearance of large single-stranded intermediates characteristic of rad52 strains. On the basis of these results, we propose that in the absence of wild-type Rad52, the interaction of RP-A with single-stranded DNA inhibits strand annealing, and that this inhibition is overcome by the rfa1-D228Y mutation.

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

2020 ◽  
Author(s):  
Danielle N. Gallagher ◽  
Nhung Pham ◽  
Annie M. Tsai ◽  
Abigail N. Janto ◽  
Jihyun Choi ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Gene Editing ◽  
Fold Increase ◽  
Single Strand ◽  
Critical Function ◽  
Strand Breaks ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Strand Annealing

AbstractThe 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 facilitate 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 in two different assays. Gene editing events were carried out either by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs) or by using a bacterial retron system that produces ssDNA templates in vivo in combination with DSBs created by Cas9. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but not Rad51, Rad54 or Rad55. Srs2 acts to prevent overloading of Rad51 on the ssDNA filament, whereas Rad59 appears to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity; thus, deletion of RAD51 suppresses both the srs2Δ and rad59Δ phenotypes. This same suppression by rad51Δ of rad59Δ is found in another DSB repair pathway, single strand annealing (SSA). In contrast, gene targeting using an 80-bp dsDNA template of the same sequence is Rad51-dependent. We also examined SSTR events in which the ssODN carried several mismatches. In the absence of the mismatch repair protein, Msh2, we found that the fate of mismatches carried on the ssDNA template are very different at the 3’ end, which can anneal directly to the resected DSB end, compared to the 5’ end. We also find that DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch close to the 3’ end of the template, similar to its removal of heterologies close to the 3’ invading end of the DSB. We further report that SSTR is accompanied by a 600-fold increase in mutations in a region adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

Efficient Repair of DNA Breaks in Drosophila: Evidence for Single-Strand Annealing and Competition With Other Repair Pathways

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 711-720
Author(s):  
Christine R Preston ◽  
William Engels ◽  
Carlos Flores
Keyword(s):  
Gene Conversion ◽  
Germ Line ◽  
Direct Repeat ◽  
Strand Break ◽  
Single Strand ◽  
P Element ◽  
Dna Breaks ◽  
Single Strand Annealing ◽  
Repair Pathways ◽  
Strand Annealing

Abstract We show evidence that DNA double-strand breaks induced in the Drosophila germ line can be repaired very efficiently by the single-strand annealing (SSA) mechanism. A double-strand break was made between two copies of a 1290-bp direct repeat by mobilizing a P transposon. In >80% of the progeny that acquired this chromosome, repair resulted in loss of the P element and loss of one copy of the repeat, as observed in SSA. The frequency of this repair was much greater than seen for gene conversion using an allelic template, which is only ∼7%. A similar structure, but with a smaller duplication of only 158 bp, also yielded SSA-like repair events, but at a reduced frequency, and gave rise to some products by repair pathways other than SSA. The 1290-bp repeats carried two sequence polymorphisms that were examined in the products. The allele nearest to a nick in the putative heteroduplex intermediate was lost most often. This bias is predicted by the SSA model, although other models could account for it. We conclude that SSA is the preferred repair pathway in Drosophila for DNA breaks between sequence repeats, and it competes with gene conversion by the synthesis-dependent strand annealing (SDSA) pathway.

scholarly journals Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated.

1992 ◽  
Vol 12 (3) ◽  
pp. 1292-1303 ◽  
Author(s):  
J Fishman-Lobell ◽  
N Rudin ◽  
J E Haber
Keyword(s):  
Direct Repeat ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Strand Breaks ◽  
Alternative Pathways ◽  
Deletion Formation ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Competing Pathways

HO endonuclease-induced double-strand breaks in Saccharomyces cerevisiae can undergo recombination by two distinct and competing pathways. In a plasmid containing a direct repeat, in which one repeat is interrupted by an HO endonuclease cut site, gap repair yields gene conversions while single-strand annealing produces deletions. Consistent with predictions of the single-strand annealing mechanism, deletion formation is not accompanied by the formation of a reciprocal recombination product. Deletions are delayed 60 min when the distance separating the repeats is increased by 4.4 kb. Moreover, the rate of deletion formation corresponds to the time at which complementary regions become single stranded. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells.

scholarly journals Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences

PLoS Genetics ◽  
2018 ◽  
Vol 14 (8) ◽  
pp. e1007543 ◽  
Author(s):  
Sreejith Ramakrishnan ◽  
Zachary Kockler ◽  
Robert Evans ◽  
Brandon D. Downing ◽  
Anna Malkova
Keyword(s):  
Single Strand ◽  
Dna Repeats ◽  
Single Strand Annealing ◽  
Pathway Choice ◽  
Strand Annealing

scholarly journals Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 693-704 ◽  
Author(s):  
Evgeny L Ivanov ◽  
Neal Sugawara ◽  
Jacqueline Fishman-Lobell ◽  
James E Haber
Keyword(s):  
Gene Conversion ◽  
Direct Repeat ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Chromosomal Dna ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Kinetics Of

Abstract HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.

scholarly journals Aberrant Double-Strand Break Repair inrad51 Mutants of Saccharomyces cerevisiae

2000 ◽  
Vol 20 (24) ◽  
pp. 9162-9172 ◽  
Author(s):  
Leslie E. Kang ◽  
Lorraine S. Symington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Double Strand Break Repair ◽  
Inverted Repeats ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Break Repair ◽  
Strand Annealing

ABSTRACT A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model forRAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required forRAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient inrad1 rad51 double mutants than in rad1 andrad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.

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