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

Publication ,  Journal Article
Gamble, D; Shaltz, S; Jinks-Robertson, S
Published in: G3 (Bethesda)
October 5, 2020

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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Published In

G3 (Bethesda)

DOI

EISSN

2160-1836

Publication Date

October 5, 2020

Volume

10

Issue

10

Start / End Page

3821 / 3829

Location

England

Related Subject Headings

  • Saccharomyces cerevisiae Proteins
  • Saccharomyces cerevisiae
  • Recombination, Genetic
  • Endonucleases
  • DNA Repair
  • DNA Breaks, Double-Stranded
  • 4905 Statistics
  • 3105 Genetics
  • 3101 Biochemistry and cell biology
  • 0604 Genetics
 

Citation

APA
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ICMJE
MLA
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Gamble, D., Shaltz, S., & Jinks-Robertson, S. (2020). Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast. G3 (Bethesda), 10(10), 3821–3829. https://doi.org/10.1534/g3.120.401603
Gamble, Dionna, Samantha Shaltz, and Sue Jinks-Robertson. “Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.G3 (Bethesda) 10, no. 10 (October 5, 2020): 3821–29. https://doi.org/10.1534/g3.120.401603.
Gamble D, Shaltz S, Jinks-Robertson S. Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast. G3 (Bethesda). 2020 Oct 5;10(10):3821–9.
Gamble, Dionna, et al. “Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.G3 (Bethesda), vol. 10, no. 10, Oct. 2020, pp. 3821–29. Pubmed, doi:10.1534/g3.120.401603.
Gamble D, Shaltz S, Jinks-Robertson S. Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast. G3 (Bethesda). 2020 Oct 5;10(10):3821–3829.

Published In

G3 (Bethesda)

DOI

EISSN

2160-1836

Publication Date

October 5, 2020

Volume

10

Issue

10

Start / End Page

3821 / 3829

Location

England

Related Subject Headings

  • Saccharomyces cerevisiae Proteins
  • Saccharomyces cerevisiae
  • Recombination, Genetic
  • Endonucleases
  • DNA Repair
  • DNA Breaks, Double-Stranded
  • 4905 Statistics
  • 3105 Genetics
  • 3101 Biochemistry and cell biology
  • 0604 Genetics