Journal
DNA REPAIR
Volume 10, Issue 6, Pages 567-576Publisher
ELSEVIER
DOI: 10.1016/j.dnarep.2011.03.003
Keywords
DNA repair; Recombination; DNA synthesis; Replication; Mph1; Srs2
Categories
Funding
- Wellcome Trust [WT076476]
- Czech Science Foundation [GACR 301/09/1917, GACR 203/09/H046]
- Ministry of Education, Youth and Sport of the Czech Republic [ME 10048, MSMT0021622413, LC06030]
- Mendel Center for Education in Biology, Biomedicine and Bioinformatics [CZ.1.07/2.3.00/09.0186]
- FNUSA-ICRC [CZ.1.05/1.1.00/02.0123]
- Hungarian Science Foundation [OTKA 77495, TAMOP-4.2.2/08/1]
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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 delta and Pol eta to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol delta and Pol eta 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. (C) 2011 Elsevier B.V. All rights reserved.
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