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Mol Cell. 2019 Jul 11;75(1):145-153.e5. doi: 10.1016/j.molcel.2019.05.005. Epub 2019 May 29.

RPA Phosphorylation Inhibits DNA Resection.

Author information

1
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA.
2
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA; Howard Hughes Medical Institute, The University of Texas at Austin, Austin, TX 78712, USA.
3
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address: ifinkelstein@cm.utexas.edu.

Abstract

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom's syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR.

KEYWORDS:

BLM; DNA repair; DNA2; EXO1; RPA; double-strand break; single-molecule

PMID:
31153714
PMCID:
PMC6625828
[Available on 2020-07-11]
DOI:
10.1016/j.molcel.2019.05.005

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