Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination.
Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.
Shiomi
Naoko
N
Radgenomics Research Group, Research Center for Charged Particle Therapy, Chiba 263-8555, Japan.
Mori
Masahiko
M
Tsuji
Hideo
H
Imai
Takashi
T
Inoue
Hirokazu
H
Tateishi
Satoshi
S
Yamaizumi
Masaru
M
Shiomi
Tadahiro
T
D002166:Camptothecin / Q000633:toxicity; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D020552:Comet Assay; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D018390:Gene Targeting; D005804:Genes, Lethal; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D016196:S Phase; D059004:Topoisomerase I Inhibitors; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014965:X-Rays
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; D059004:Topoisomerase I Inhibitors; D025801:Ubiquitin; C101467:XRCC4 protein, human; D044767:Ubiquitin-Protein Ligases; D002166:Camptothecin