Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities

Marek Sebesta, Peter Burkovics, L. Haracska, Lumir Krejci

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

The error-free repair of double-strand DNA breaks by homologous recombination (HR) ensures genomic stability using undamaged homologous sequence to copy genetic information. While some of the aspects of the initial steps of HR are understood, the molecular mechanisms underlying events downstream of the D-loop formation remain unclear. Therefore, we have reconstituted D-loop-based in vitro recombination-associated DNA repair synthesis assay and tested the efficacy of polymerases Pol δ and Pol η to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol δ and Pol η extended up to 50% of the D-loop substrate, but differed in product length and dependency on proliferating cell nuclear antigen (PCNA). Mph1, but not Srs2 or Sgs1, displaced the extended primer very efficiently, supporting putative role of Mph1 in promoting the synthesis-dependent strand-annealing pathway. The experimental system described here can be employed to increase our understanding of HR events following D-loop formation, as well as the regulatory mechanisms involved.

Original languageEnglish
Pages (from-to)567-576
Number of pages10
JournalDNA Repair
Volume10
Issue number6
DOIs
Publication statusPublished - Jun 10 2011

Fingerprint

Homologous Recombination
DNA Repair
Repair
DNA
Proliferating Cell Nuclear Antigen
Assays
Double-Stranded DNA Breaks
Genomic Instability
Annealing
Sequence Homology
Genetic Recombination
Substrates
In Vitro Techniques

Keywords

  • DNA repair
  • DNA synthesis
  • Mph1
  • Recombination
  • Replication
  • Srs2

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. / Sebesta, Marek; Burkovics, Peter; Haracska, L.; Krejci, Lumir.

In: DNA Repair, Vol. 10, No. 6, 10.06.2011, p. 567-576.

Research output: Contribution to journalArticle

Sebesta, Marek ; Burkovics, Peter ; Haracska, L. ; Krejci, Lumir. / Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. In: DNA Repair. 2011 ; Vol. 10, No. 6. pp. 567-576.
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