Visualization of Recombination-Mediated Damage Bypass by Template Switching

Nat Struct Mol Biol. 2014 Oct;21(10):884-92. doi: 10.1038/nsmb.2888. Epub 2014 Sep 7.

Abstract

Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase δ-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatids
  • DNA Damage*
  • DNA Polymerase III / genetics
  • DNA Repair*
  • DNA Replication / genetics*
  • DNA, Cruciform
  • DNA, Fungal / biosynthesis
  • DNA, Fungal / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Recombination, Genetic*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics
  • Templates, Genetic*
  • Ubiquitination

Substances

  • DNA, Cruciform
  • DNA, Fungal
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • DNA Polymerase III