Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair |
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Affiliation: | 2. Co-Innovation Center for Marine Bio-Industry Technology of Jiangsu Province, Lianyungang, Jiangsu 222005, PR China;1. Université Claude Bernard Lyon1, F-69622 Villeurbanne, France;2. INSERM, U865, IFR62, F-69 372 Lyon, France;3. Cancer Research Center of Lyon, UMR 1052, INSERM, 69 372, Lyon cedex 08, France;4. Department of Urology, Nijmegen Medical Centre, Radboud University, Nijmegen, The Netherlands |
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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 4 h. However, RPA did not reach its maximum accumulation level until 3 h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8 h, 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. |
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Keywords: | RPA DNA replication DNA repair Replication stress Nucleotide excision repair DNA damage |
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