| Abstract
| - The key step in skin cancer induction by UV light is thought to bethe mutagenic DNA synthesispast a DNA photoproduct in a proto-oncogene or tumor suppressor gene.To investigate this critical step,we have constructed an SV40 vector containing a cis-syn thymine dimer,the major DNA photoproductinduced by UVB light, within an AseI site at a location thatwould initially be replicated by leadingstrand synthesis. When the dimer-containing SV40 vector wasincubated with cell-free HeLa extracts inthe presence of TAg, and then digested with AseI, a 2325 bpfragment corresponding to inhibition ofcleavage at the dimer site was observed, suggesting that the dimer hadterminated synthesis and/or hadbeen bypassed. When the reaction was limited to one round ofreplication and the products of restrictionenzyme digestion were examined by denaturing gel electrophoresis, bandscorresponding to both terminationand bypass were observed in roughly a one-to-one ratio. Whereasincreasing the dNTP concentrationfrom 10 μM to 1 mM increased the ratio of bypass to termination from0.6 to 2.6, it had no effect on thesite of termination, which occurred exclusively one nucleotide beforethe dimer. Experiments in whichdGTP was held constant at 25 μM and various combinations of theremaining nucleotides were raisedfrom 25 μM to 1 mM showed substantial increases in thebypass-to-termination ratio, with the greatesteffect seen for raising all three nucleotides to 1 mM. Replicationby primary fibroblast XPV extracts wasalso investigated and found to be greatly stimulated by rhRPA, whereasthe stimulatory effect for HeLacell extracts was variable. In the presence of rhRPA, the XPVextracts were also found to bypass thecis-syn dimer, which contrasts with a recent report that could notdetect dimer bypass in SV40 transformedXPV extracts in the absence of added replication factors[Cordeiro-Stone, M., et al. (1997) J.Biol.Chem.272, 13945−13954].
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