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Geneious prime citation3/7/2023 We examined the tolerance of differently distributed mismatches on a homologous donor template. We used budding yeast Saccharomyces cerevisiae to investigate Rad51-mediated BIR, where the invading 3’ end of the DSB and a donor template share 108 bp of homology. Break-induced replication (BIR) is a homologous recombination pathway that results in a nonreciprocal translocation of chromosome ends. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3’ end of the donor.ĭNA double-strand breaks (DSBs) are the most lethal forms of DNA damage and inaccurate repair of these breaks presents a serious threat to genomic integrity and cell viability. These data suggest that DNA polymerase δ “chews back” the 3’ end of the invading strand without any mismatch-dependent cues from the strand invasion structure. Surprisingly, the probability of a mismatch 27 nt from the 3’ end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6 th base or fully homologous. Mismatch incorporation depends on the 3’→ 5’ proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3’ end. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Now, using substrates of either 90 or 108 nt–the latter being the size of the in vivo templates–we find that in vitro D-loop results are very similar to the in vivo results. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6 th base was mismatched. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed but mismatch position-specific effects are also important. A donor with all 10 mismatches clustered at the 3’ invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5’ end. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. ![]() Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. BIR still occurs about 10% as often when every 6 th base is mismatched as with a perfectly matched donor. You must submit a request through “ NCI at Your Service” to obtain access to Geneious Pro Software.Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3’ end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. Sequencing & multiplex primers (coming soon).Import chromatograms & attach to plates.Velvet, Maq, Bowtie on Geneious Server™.Geneious is a DNA, RNA, and protein sequence alignment, assembly, and analysis software platform, integrating bioinformatics and molecular biology tools into a simple interface.
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