A Proximity Ligation-Based Method for Quantitative Measurement of D-Loop Extension in S. cerevisiae

Methods Enzymol. 2018:601:27-44. doi: 10.1016/bs.mie.2017.11.024. Epub 2018 Feb 24.

Abstract

Homologous recombination faithfully restores the sequence information interrupted by a DNA double-strand break by referencing an intact DNA molecule as a template for repair DNA synthesis. DNA synthesis is primed from 3'-OH end of the invading DNA strand in the displacement loop (D-loop). Here, we describe a simple and quantitative proximity ligation-based assay to study the initiation of homologous recombination-associated DNA synthesis initiated at the D-loop and final product formation. The D-loop extension assay overcomes the semiquantitative nature and some limitations of the current PCR-based technique and facilitates the study of the recombination-associated DNA synthesis.

Keywords: D-loop; DNA synthesis; DSB repair; Homologous recombination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Breaks, Single-Stranded*
  • DNA Ligases / metabolism*
  • DNA, Fungal / metabolism*
  • Genetic Techniques*
  • Recombinational DNA Repair*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*

Substances

  • DNA, Fungal
  • DNA Ligases