Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: the role of DNA polymerase kappa

J Biol Chem. 2004 Dec 17;279(51):53298-305. doi: 10.1074/jbc.M409155200. Epub 2004 Oct 9.


Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK(+/+) mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 +/- 4%, which is an order of magnitude higher than in Escherichia coli. In PolK(-/-) MEFs, bypass was 16 +/- 1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase kappa (polkappa). In contrast, poleta was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking polkappa. Expression of polkappa from a plasmid introduced into PolK(-/-) MEFs restored both the extent and fidelity of bypass across BP-G. Polkappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polkappa and poleta, suggesting that the biological role of polkappa in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that polkappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Benzo(a)pyrene / metabolism*
  • Cell Line
  • Cells, Cultured
  • DNA / biosynthesis*
  • DNA / metabolism
  • DNA Adducts*
  • DNA Damage*
  • DNA Primers / genetics
  • DNA Repair* / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / metabolism
  • Fibroblasts / metabolism
  • Humans
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • Mutagenicity Tests
  • Mutation
  • Nicotiana
  • Plasmids / metabolism
  • Time Factors
  • Transgenes
  • Ultraviolet Rays


  • DNA Adducts
  • DNA Primers
  • Benzo(a)pyrene
  • DNA
  • DNA-Directed DNA Polymerase
  • Rad30 protein