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

Biochemistry. 1997 Aug 5;36(31):9486-92. doi: 10.1021/bi970650o.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage M13 / genetics*
  • Cell Extracts
  • DNA Polymerase I / metabolism
  • DNA Polymerase II / metabolism
  • DNA Replication*
  • DNA, Single-Stranded / biosynthesis
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Mutagenesis, Site-Directed
  • RNA, Viral / biosynthesis*
  • SOS Response, Genetics*


  • Cell Extracts
  • DNA, Single-Stranded
  • RNA, Viral
  • DNA Polymerase I
  • DNA Polymerase II