RELATIVE PROBABILITY OF MUTAGENIC TRANSLESION SYNTHESIS ON THE LEADING AND LAGGING STRANDS DURING REPLICATION OF UV-IRRADIATED DNA IN A HUMAN CELL EXTRACT

We have previously demonstrated mutagenic bypass of pyrimidine dimers during SV40 origin-dependent replication of UV-irradiated DNA in human cell extracts [Thomas, D. C., & Kunkel, T. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7744-7748]. Here we use two vectors having the origin of replication on opposite sides of a lacZalpha reporter gene to examine the relative probability of mutagenic translesion synthesis on the leading and lagging strands. Although replication of both vectors is inhibited by UVB irradiation in a dose-dependent manner, the covalently closed DNA products of replication contain T4 endonuclease sensitive sites, indicating that bypass of cyclobutane pyrimidine dimers occurred. At fluences of 70 and 100 J/m2, the mutant frequencies obtained with both vectors are substantially higher than with control DNAs. Sequence analysis of mutants obtained with both vectors reveal three types of mutations at frequencies significantly above those obtained from replication of undamaged DNA. These are C --> T transitions, accounting for about two-thirds of the mutants, a small number of CC --> TT substitutions, and complex mutations. Comparing the distribution of C --> T substitutions in the two spectra permits an estimation of the probability of mutagenic translesion replication of the same sequence when replicated as the leading or lagging strand. The data suggest that the overall average UV-independent C --> T substitution probability per phenotypically detectable dipyrimidine site is the same during leading and lagging strand replication. However, statistically significant differences are observed when the distribution of C -> T substitutions is considered. The substitution probability at some positions is higher when replicated as the lagging strand than when replicated as the leading strand, while at other sites the opposite is observed. Thus the fidelity of leading and lagging strand translesion synthesis varies by position.