[Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells].
Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.
Nikiforov
A A
AA
Institute of Cytology RAS, St. Petersburg, Russia, Free University of Berlin, Germany. nikiforovan@hotmail.com
Svetlova
M P
MP
Solov'eva
L V
LV
Ziegler
M
M
Oei
S
S
Nikolaishvili-Feinberg
N
N
Codeiro-Stone
M
M
Tomilin
N V
NV
D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008322:Mammals; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases; D000099270:Y-Family DNA Polymerases
D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D008741:Methyl Methanesulfonate; D018809:Proliferating Cell Nuclear Antigen; D019311:Staurosporine; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases