title: DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation.
abstract: Rad18 protein is required for mono-ubiquitination of PCNA and trans-lesion synthesis during DNA lesion bypass in eukaryotic cells but it remains unknown how it is activated after DNA damage. We expressed GFP-tagged human (h)Rad18 in Chinese hamster cells and found that it can be completely extracted from undamaged nuclei by Triton X-100 and methanol. However, several hours after treatment with methyl methanesulfonate (MMS) Triton-insoluble form of GFP-hRad18 accumulates in S-phase nuclei where it colocalizes with PCNA. This accumulation is suppressed by inhibitors of protein kinases staurosporine and wortmannin but is not effected by roscovitine. We also found that methyl methanesulfonate induces phosphorylation of Ser-317 in protein kinase Chk1 and Ser-139 in histone H2AX and stimulates formation of single-stranded DNA at replication foci. Together, our results suggest that MMS-induced accumulation of hRad18 protein at stalled forks involves protein phosphorylation which may be performed by S-phase checkpoint kinases.
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, Tikchoretskii Av. 4, 194064 St. Petersburg, Russia.
authors.forename: Andrey
authors.initials: A
authors.lastname: Nikiforov
authors.forename: Maria
authors.initials: M
authors.lastname: Svetlova
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Solovjeva
authors.forename: Lioudmila
authors.initials: L
authors.lastname: Sasina
authors.forename: Joseph
authors.initials: J
authors.lastname: Siino
authors.forename: Igor
authors.initials: I
authors.lastname: Nazarov
authors.forename: Morton
authors.initials: M
authors.lastname: Bradbury
authors.forename: Nikolai
authors.initials: N
authors.lastname: Tomilin
mesh_terms: D000818:Animals; D006224:Cricetinae; D004247:DNA / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology*; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D009153:Mutagens / Q000494:pharmacology; D010766:Phosphorylation; D044767:Ubiquitin-Protein Ligases
chemical_list: D004268:DNA-Binding Proteins; D009153:Mutagens; C485954:RAD18 protein, human; D004247:DNA; D008741:Methyl Methanesulfonate; D044767:Ubiquitin-Protein Ligases