Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans

DNA Repair (Amst). 2013 Sep;12(9):691-8. doi: 10.1016/j.dnarep.2013.05.001. Epub 2013 May 31.

Abstract

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.

Keywords: D-loop; DNA repair synthesis; Homologous recombination; Reconstitution; TLS polymerases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Damage
  • DNA Polymerase III / chemistry
  • DNA Polymerase III / physiology
  • DNA Replication
  • DNA, Single-Stranded / biosynthesis
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / physiology
  • HeLa Cells
  • Homologous Recombination*
  • Humans
  • Osmolar Concentration
  • Proliferating Cell Nuclear Antigen / chemistry*
  • Proliferating Cell Nuclear Antigen / physiology
  • RNA-Binding Protein FUS / chemistry
  • RNA-Binding Protein FUS / physiology
  • Rad51 Recombinase / chemistry

Substances

  • DNA, Single-Stranded
  • Proliferating Cell Nuclear Antigen
  • RNA-Binding Protein FUS
  • DNA polymerase iota
  • RAD51 protein, human
  • Rad51 Recombinase
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Rad30 protein