Abstract
Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.
Highlights
The presence of short stretches of overlapping sequence is a frequent feature of pathogenic chromosomal translocation breakpoints in human cells and has been implicated in juxtaposing two DNA ends for the error-prone repair of DNA breaks in both yeast and vertebrates [1,2,3]
The prevalence of 2–20 bp of imperfect overlapping sequences (a.k.a. microhomology [MH]) at the breakpoint junctions suggests the type of repair events joining two chromosomal fragments and the formation of oncogenic chromosomal translocations
We discovered that MH-mediated end joining (MMEJ) operates with kinetics markedly slower than other repair options
Summary
The presence of short stretches of overlapping sequence (microhomology, MH) is a frequent feature of pathogenic chromosomal translocation breakpoints in human cells and has been implicated in juxtaposing two DNA ends for the error-prone repair of DNA breaks in both yeast and vertebrates [1,2,3]. This so-called microhomology-mediated end joining (MMEJ) is genetically distinct from Ku-dependent classical end joining or homologous recombination and becomes a prominent repair option when conventional repair mechanisms become inactivated or unavailable. Resection and the formation of ssDNA are key steps in MMEJ and likely dictate the types of repair outcomes and chromosomal integrity upon DNA breakage
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