Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.

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2020-10

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Abstract

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' vs. 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' vs. 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

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10.1534/g3.120.401603

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Gamble, Dionna, Samantha Shaltz and Sue Jinks-Robertson (2020). Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast. G3 (Bethesda, Md.), 10(10). pp. 3821–3829. 10.1534/g3.120.401603 Retrieved from https://hdl.handle.net/10161/30372.

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Scholars@Duke

Shaltz

Samantha Shaltz

Lab Research Analyst II
Jinks-Robertson

Sue Jinks-Robertson

James B. Duke Distinguished Professor Emerita

My research focuses on the regulation of genetic stability and primarily uses budding yeast (Saccharomyces cerevisiae) as a model genetic system.  The two primary research goals in the budding yeast system are (1) defining molecular structures and mechanisms of mitotic recombination intermediates and (2) understanding how and why transcription destabilizes the underlying DNA template.  We also have initiated studies of mutagenesis in the pathogenic fungus Cryptococcus neoformans.  We have found that a shift to the human body temperature mobilizes transposable elements, and suggest that this promotes rapid adaptation to the harsh host environment.  


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