| We are examining the molecular mechanisms of mutation induction by carcinogens, in order to reduce the incidence of cancer associated with such agents. In the yeast Saccharomyces cerevisiae, the ubiquitin ligase rad18 has been shown to be required for the successful replication of DNA past stalled replication forks after UV exposure. This protein has been extensively studied in yeast, but recently the human homolog has been discovered. Acting in conjunction with the ubiquitin-conjugating enzyme rad6, rad18 binds to sites of single-stranded DNA at sites of DNA damage by an unknown signaling mechanism. This complex acts to monoubiquitinate proliferating cell nuclear antigen (PCNA) at lysine residue 164 (K164). We hypothesize that RAD18 in humans (hRAD18) initiates the signal that recruits mutagenic translesion polymerases to the sites of stalled replication forks. To examine the rote of hRAD18, we established cell lines that have reduced levels of hRAD18 by virtue of the stable expression of ribozymes or antisense to the transcript. We found that an 85% reduction in the level of protein reduced the frequency of mutations in the HPRT gene by UV254nm or benzo[a]pyrenediolepoxide (BPDE) virtually to background levels. Using immunohistochemistry, we determined that DNA damage induced by UV or BPDE causes hRAD18 to accumulate in the nucleus in a focal pattern, although protein levels of hRAD18 remain constant as indicated by Western analysis. These foci co-localize with PCNA, the clamp protein present in DNA replication forks only when damaged in S phase of the cell cycle. These experimental data support the hypothesis that RAD18 is involved in the signaling of mutagenic translesion polymerases. |
