Role of high-fidelity Escherichia coli DNA polymerase I in replication bypass of a deoxyadenosine DNA-peptide cross-link

J Bacteriol. 2011 Aug;193(15):3815-21. doi: 10.1128/JB.01550-10. Epub 2011 May 27.

Abstract

Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (N⁶-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past N⁶-dA-linked peptides. Based on these results, we hypothesized that another member of that family, Escherichia coli polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific N⁶-dA dodecylpeptide cross-link were created and utilized for in vitro DNA replication assays using E. coli DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using E. coli strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific N⁶-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past N⁶-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.

Publication types

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

MeSH terms

  • Cross-Linking Reagents / chemistry
  • DNA Adducts / chemistry
  • DNA Adducts / genetics
  • DNA Damage
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA Repair
  • DNA Replication*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics*
  • DNA, Single-Stranded / genetics
  • Deoxyadenosines / chemistry
  • Deoxyadenosines / genetics*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Peptides / chemistry*

Substances

  • Cross-Linking Reagents
  • DNA Adducts
  • DNA, Bacterial
  • DNA, Single-Stranded
  • Deoxyadenosines
  • Escherichia coli Proteins
  • Peptides
  • DNA Polymerase I