Abstract
ABSTRACTAlthough gene conversion (GC) inSaccharomyces cerevisiaeis the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially-copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage atMATα, repaired by anHMR::Kl-URA3donor. We inserted intoHMR::Kl-URA3an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yieldmat::Kl-ura3::QP+4(Ura-) outcomes, but template-switching produces Ura+colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP+4 arm is encountered first, Ura+derivatives only occur after second-end capture and second-strand synthesis. QP+4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (pol3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a +1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura3+recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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