The role of specific amino acid residues in the active site of Escherichia coli DNA polymerase I on translesion DNA synthesis across from and past an N-2-aminofluorene adduct

Biochemistry. 2007 Mar 13;46(10):2599-607. doi: 10.1021/bi061324o. Epub 2007 Feb 17.


Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. A central part of these studies is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template. To address the importance of polymerase architecture on replication across from this type of bulky DNA adduct, three active-site mutants of Escherichia coli DNA polymerase I (Klenow fragment) were used to study DNA synthesis on DNA modified with the carcinogen N-2-aminofluorene (AF). Running-start synthesis studies showed that full-length synthesis past the AF adduct was inhibited for all of the mutants, but that this inhibition was substantially less for the F762A mutant. Single nucleotide extension and steady-state kinetic experiments showed that the Y766S mutant displayed higher rates of insertion of each incorrect nucleotide relative to WT across from the dG-AF adduct. This effect was not observed for F762A or E710A mutants. Similar experiments that measured synthesis one nucleotide past the dG-AF adduct revealed an enhanced preference by the F762A mutant for dG opposite the T at this position. Finally, synthesis at the +1 and +2 positions was inhibited to a greater extent for the Y766S and E710A mutants compared with both the WT and F762A mutants. Taken together, this work is consistent with the model that polymerase geometry plays a crucial role in both the insertion and extension steps during replication across from bulky DNA lesions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 2-Acetylaminofluorene / chemistry*
  • Binding Sites
  • DNA / biosynthesis*
  • DNA Adducts / metabolism
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*


  • DNA Adducts
  • DNA
  • 2-Acetylaminofluorene
  • DNA Polymerase I