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-induced Escherichia coli, we have developed a high-resolution DNA replication system based on E. coli cell extracts and M13 genomic DNA templates 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 permits one to quantitatively measure the efficiency of translesion synthesis. Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion. In contrast, the efficiency of translesion synthesis across SOS-independent lesions such as O6-methylguanine and DNA uracil is around 90%, very close to the values obtained for control DNA templates. The efficiency of translesion synthesis across 3,N4-ethenocytosine and 1, N6-ethenoadenine is around 20%, a value that is similar to the in vivo efficiency deduced from the effect of the lesions on the survival of transfected M13 ssDNA. Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essentially similar results are obtained with extracts from polA- or polB-defective cells. The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions included in this study suggest that the assay may be suitable for modeling mutagenesis in an accessible in vitro environment.
Wang
G
G
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, MSB F607, Newark, New Jersey 07103-2714, USA.
Rahman
M S
MS
Humayun
M Z
MZ
D017104:Bacteriophage M13 / Q000235:genetics*; D002457:Cell Extracts; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000096:biosynthesis*; D013014:SOS Response, Genetics
D002457:Cell Extracts; D004277:DNA, Single-Stranded; D012367:RNA, Viral; D004256:DNA Polymerase I; D004257:DNA Polymerase II