title: Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair.
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.
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Audrey M
authors.initials: AM
authors.lastname: Gourdin
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Loes
authors.initials: L
authors.lastname: van Cuijk
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
authors.forename: Maria
authors.initials: M
authors.lastname: Tresini
authors.affiliation: 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.
authors.forename: Martijn S
authors.initials: MS
authors.lastname: Luijsterburg
authors.affiliation: Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
authors.forename: Alex L
authors.initials: AL
authors.lastname: Nigg
authors.affiliation: CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France.
authors.forename: Guiseppina
authors.initials: G
authors.lastname: Giglia-Mari
authors.affiliation: Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Electronic address: W.Vermeulen@erasmusmc.nl.
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
authors.affiliation: Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Electronic address: J.Marteijn@erasmusmc.nl.
authors.forename: Jurgen A
authors.initials: JA
authors.lastname: Marteijn
mesh_terms: D002453:Cell Cycle; D002460:Cell Line / Q000528:radiation effects; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D051818:Replication Protein C / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C081632:RFC1 protein, human; C497167:RPA1 protein, human; D051716:Replication Protein A; C095888:xeroderma pigmentosum group F protein; D049452:Green Fluorescent Proteins; D051818:Replication Protein C
keywords: DNA damage; DNA repair; DNA replication; Nucleotide excision repair; RPA; Replication stress