REPLICATION OF M13 SINGLE-STRANDED VIRAL-DNA BEARING SINGLE SITE-SPECIFIC ADDUCTS BY ESCHERICHIA-COLI CELL-EXTRACTS - DIFFERENTIAL EFFICIENCY OF TRANSLESION DNA-SYNTHESIS FOR SOS-DEPENDENT AND SOS-INDEPENDENT LESIONS

In Order to characterize mutagenic translesion DNA synthesis in UVM-in duced Escherichia coli, we have developed a high-resolution DNA replic ation system based on E. coli cell extracts and M13 genomic DNA templa tes bearing mutagenic lesions, The assay is based on the conversion of M13 viral single-stranded DNA (ssDNA) bearing a single site-specific DNA lesion to the double-stranded replicative form (RF) DNA, and permi ts one to quantitatively measure the efficiency of translesion synthes is. Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion. In c ontrast, the efficiency of translesion synthesis across SOS-independen t lesions such as O-6-methylguanine and DNA uracil, is around 90%, ver y close to the values obtained for control DNA templates. The efficien cy of translesion synthesis across 3,N-4-ethenocytosine and 1,N-6-ethe noadenine is around 20%, a value that is similar to the in vivo effici ency deduced from the effect of the lesions on the survival of transfe cted M13 ssDNA. Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essenti ally similar results are obtained with extracts from polA- or polB-def ective cells. The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions inc luded in this study suggest that the assay may be suitable for modelin g mutagenesis in an accessible in vitro environment.