Dynamic targeting of the replication machinery to sites of DNA damage.
Components of the DNA replication machinery localize into discrete subnuclear foci after DNA damage, where they play requisite functions in repair processes. Here, we find that the replication factors proliferating cell nuclear antigen (PCNA) and RPAp34 dynamically exchange at these repair foci with discrete kinetics, and this behavior is distinct from kinetics during DNA replication. Posttranslational modification is hypothesized to target specific proteins for repair, and we find that accumulation and stability of PCNA at sites of damage requires monoubiquitination. Contrary to the popular notion that phosphorylation on the NH2 terminus of RPAp34 directs the protein for repair, we demonstrate that phosphorylation by DNA-dependent protein kinase enhances RPAp34 turnover at repair foci. Together, these findings support a dynamic exchange model in which multiple repair factors regulated by specific modifications have access to and rapidly turn over at sites of DNA damage.
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Ave., Cincinnati, OH 45267, USA. das67@georgetown.edu
David A
DA
Solomon
M Cristina
MC
Cardoso
Erik S
ES
Knudsen
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D014466:Ultraviolet Rays
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; C490673:RPAp34 protein, rat; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D004247:DNA; D001973:Bromodeoxyuridine; D008239:Lysine; D002945:Cisplatin