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Mol Cell. 2017 Sep 7;67(5):867-881.e7. doi: 10.1016/j.molcel.2017.07.001. Epub 2017 Jul 27.

Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

Author information

1
DNA Metabolism Laboratory, IFOM, FIRC Institute for Molecular Oncology, 20139 Milan, Italy.
2
Department of Biology, Masaryk University, Brno 625 00, Czech Republic.
3
Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK.
4
Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA.
5
Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1GA, UK. Electronic address: lp212@cam.ac.uk.
6
Department of Biology, Masaryk University, Brno 625 00, Czech Republic; National Centre for Biomolecular Research, Masaryk University, Brno 625 00, Czech Republic; International Clinical Research Center, St. Anne's University Hospital, Brno 656 91, Czech Republic. Electronic address: lkrejci@chemi.muni.cz.
7
DNA Metabolism Laboratory, IFOM, FIRC Institute for Molecular Oncology, 20139 Milan, Italy. Electronic address: vincenzo.costanzo@ifom.eu.

Abstract

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

KEYWORDS:

Brca2; DNA replication; Mre11; Rad51; Xenopus laevis; fork protection

PMID:
28757209
PMCID:
PMC5594205
DOI:
10.1016/j.molcel.2017.07.001
[Indexed for MEDLINE]
Free PMC Article

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