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DNA Repair (Amst). 2014 Dec;24:46-56. doi: 10.1016/j.dnarep.2014.09.013. Epub 2014 Oct 16.

Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair.

Author information

1
Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
2
Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands; Swammerdam Institute for Life Sciences, University of Amsterdam, Sciencepark 904, 1098 XH Amsterdam, The Netherlands.
3
Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
4
CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France.
5
Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Electronic address: W.Vermeulen@erasmusmc.nl.
6
Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Electronic address: J.Marteijn@erasmusmc.nl.

Abstract

The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.

KEYWORDS:

DNA damage; DNA repair; DNA replication; Nucleotide excision repair; RPA; Replication stress

PMID:
25453469
DOI:
10.1016/j.dnarep.2014.09.013
[Indexed for MEDLINE]

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