Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells

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 κ (polκ). In contrast, polη 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 polκ. Expression of polκ from a plasmid introduced into PolK–/– MEFs restored both the extent and fidelity of bypass across BP-G. Polκ was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polκ and polη, suggesting that the biological role of polκ 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 polκ bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases. 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 κ (polκ). In contrast, polη 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 polκ. Expression of polκ from a plasmid introduced into PolK–/– MEFs restored both the extent and fidelity of bypass across BP-G. Polκ was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polκ and polη, suggesting that the biological role of polκ 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 polκ bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases. Genomic DNA is constantly subject to damage caused by both external agents, such as sunlight, and endogenous chemicals, such as reactive oxygen species. Most of this damage is eliminated by error-free DNA repair mechanisms, thereby restoring the DNA to its native sequence (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995Google Scholar). However, a significant number of lesions escape repair and might therefore interfere with DNA replication and gene expression. Such interference can be mitigated by DNA damage tolerance mechanisms, primarily translesion DNA synthesis (TLS 1The abbreviations used are: TLS, translesion DNA synthesis; BP, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BP-G, (+)-trans-benzo[a]pyrene-guanine adduct; MEF, mouse embryo fibroblasts; pol, DNA polymerase; MOPS, 4-morpholinepropanesulfonic acid.; also termed translesion replication) (2Goodman M.F. Trends Biochem. Sci. 2000; 25: 189-195Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 4Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 5Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar) and postreplicative recombinational repair (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. 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This is by to and a to cells from are to and by (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995Google Scholar). human polη across with efficiency and accuracy R.E. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; PubMed Google Scholar). These that in cells in which polη is the bypass of lesions is effected by other DNA polymerases with efficiency in and fidelity in and for a in TLS in vivo also for polζ and In of the with to (14Gibbs P.E. McGregor W.G. Maher Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P.E. Wang Z. McGregor W.G. Lawrence C.W. Maher Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). In vivo showed that fibroblasts in from PolK–/– mouse are to both and by BP, to cells from PolK+/+ mouse suggesting that polκ is in bypass across BP-G adducts in vivo T. H. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). In vitro that polκ bypasses BP-G the F. X. Wang M. O. Wang Z. 2000; 28: PubMed Google Scholar, A. H. S. 2002; PubMed Scopus Google Scholar, X. D. O. Wang Z. DNA 2002; PubMed Scopus Google Scholar, X. A. X. Wang Z. S. PubMed Scopus Google Scholar). In this used a quantitative TLS assay to bypass across a BP-G adduct in mammalian cells. We that BP-G is bypassed in mammalian cells with a relatively high efficiency of an order of magnitude higher than in polκ is for at least two-thirds of lesion bypass and bypass by polκ with than bypass by other DNA polymerases. and with and and and were from and a of and for cell were from was from coli polymerase DNA and were from DNA polymerases and were from a lesion were by synthetic abasic were by the of the in were from to the plasmid recovery from mammalian adduct were generated as J. S. 16: PubMed Scopus Google Scholar). The used to the plasmids and were the of the and to using a as a The were from the and a The of the plasmids a synthetic abasic and a was N.B. G. Livneh Z. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Livneh Z. 1999; PubMed Scopus Google Scholar). The plasmids and were to that the the The plasmid is a of This plasmid was as that the plasmid used was is the of the from plasmid and a the gene from plasmid and with and The of the human polκ, was by from cell as using the for the of the and for the of and for the of and The were with and and the was from the using and and into the of plasmid to plasmid The sequence of the gene was The polκ was generated by using as a and the and The was with and and into and to the The sequence of polκ was by DNA from and mouse fibroblasts were D. C. S. Friedberg E.C. G. J. 2002; PubMed Scopus Google Scholar). The human and also L. M. W. H. 1999; Google were from A. R. of The human cells were cultured in The cells were in with was with and The cells were at in a TLS in quantitative TLS assay is a of the assay S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). The assay of mammalian cells with a plasmid with a plasmid and a plasmid is in with a gapped plasmid a lesion with the plasmid and plasmid of the transfected cells to TLS, the plasmid are and gapped plasmids that were not and are by The plasmid is into an coli and are in for plasmids that and for the The extent of bypass is by the of with the plasmid by the of with the gapped plasmid S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). In the the plasmid by a gapped plasmid a lesion thereby the TLS cells are with a plasmid the plasmid a gapped plasmid a lesion and the plasmid for and lesion the plasmids were from the cells using such that in plasmids assay the of in the plasmid was into an coli and in as in the for plasmids that and for the in plasmid TLS in this was by the of coli The the cells were with a DNA of a plasmid of a gapped plasmid lesion and of the plasmid using O. F. D. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of lesion bypass was by the number of to the plasmid recovery from mammalian cells. of by the number of of plasmids were from and the sequence the lesion was by DNA analysis in the in this In the polκ the MEFs were with a DNA of plasmid of gapped plasmid lesion and of the In this the cells were with to the plasmids were from and the sequence the lesion was by DNA analysis in the in this TLS in plasmids the BP-G adduct and and the plasmid the adduct were used in to coli cells as A. Izhar L. Livneh Z. Mol. Cell. 2002; 10: 917-924Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, N.B. G. Livneh Z. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The cells were at by a recovery of at which were in with the gapped plasmids with and the BP-G adduct using the A. Izhar L. Livneh Z. Mol. Cell. 2002; 10: 917-924Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). was by the number of with the plasmid by the number of with the gapped plasmid the lesion. In this results from are is the of cells was used for the gapped plasmids with and the and the of the results was by for of gapped plasmid The used in this were coli and coli both in In DNA used for this assay were by a to the by a was used for the was used for the both a with the the the lesion. Analysis by native revealed that of the were to the of and and DNA by coli polymerase also and by human DNA polymerase human DNA polymerase human DNA polymerase also and The were at for were by an of a of and were by which were and using a The extent of bypass was by the of bypass by the of TLS across a in than in lesion bypass assay used is a of a assay of cells in with a gapped DNA plasmid a lesion at a and an are from cells and are introduced into coli cells for and analysis in which are by TLS in the mammalian cells to in coli S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) the a gapped plasmid the lesion and a was with the plasmid the lesion. In DNA is in the plasmid The of the extent of and lesion bypass for the assay measures TLS the recovery of gapped We used gapped plasmids BP-G adducts in order to bypass in mammalian cells. were the sequence from the the M.F. A. Science. 1996; 274: PubMed Scopus Google Scholar). the sequence the sequence is with in the plasmids were and introduced into the coli the TLS was low the in this plasmid was in PolK+/+ TLS was of magnitude an extent of 48 ± results were with plasmid ± This extent of bypass is higher than for bypass across BP-G adducts in coli T. A.K. Chem. 10: PubMed Scopus Google Scholar, R. R. J. Fuchs R.P.P. EMBO J. 2000; PubMed Scopus Google Scholar, X. H. O'Donnell M. Woodgate R. Goodman M.F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the might from DNA sequence which are to bypass across BP-G adducts in vitro and in coli in vivo M. S. A. S. T. 1996; PubMed Scopus Google Scholar, R. T. PubMed Scopus Google Scholar, T. A. PubMed Scopus Google Scholar, PubMed Scopus Google TLS across the BP-G adduct in coli using the gapped plasmids used in the mammalian cells. The as A. Izhar L. Livneh Z. Mol. Cell. 2002; 10: 917-924Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, N.B. G. Livneh Z. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google of coli cells with plasmid by a the cells were in with the gapped plasmid the lesion. TLS was by the of with the plasmids to the number of with the gapped plasmid a lesion. can be in bypass across the BP-G adduct was in both with the and at caused a in bypass not bypass across the BP-G adduct was at least an order of magnitude more efficient in embryo fibroblasts than in of bypass across BP-G adducts in mouse and in coli coli number of in a assay with of the were in order to an ± ± DNA the plasmid was introduced into coli the mouse ± DNA the plasmid was introduced into coli the mouse ± coli coli used in this are in are of with the plasmid the and the gapped plasmid the in a assay with of the In order to an were to ± ± ± ± ± ± ± ± The number of in a assay with of the were in order to an DNA the plasmid was introduced into coli the mouse The coli used in this are in are of with the plasmid the and the gapped plasmid the in a assay with of the In order to an were to in a in TLS across a the role of polκ in bypass across BP-G adducts by the TLS assay in in PolK+/+ and PolK–/– cells. can be in and bypass across the BP-G adduct in gapped plasmid in PolK–/– MEFs was 16 ± 1%, than in PolK+/+ results were with plasmid In this TLS was ± and ± in PolK+/+ and PolK–/– of bypass across a BP-G adduct in and MEFs in the of polκ from a cells coli number of in a assay with of for the was used to for a efficiency of the This not the results of lesion which are by the of ± ± ± ± ± ± The number of in a assay with of for the was used to for a efficiency of the This not the results of lesion which are by the of in a In order to the TLS in PolK–/– is due to the of polκ, used a plasmid human polκ from the to the in PolK–/– cells. and the results of such with plasmid TLS in the PolK–/– cells that were transfected with the was ± However, with the plasmid human polκ, bypass to ± to the in PolK+/+ cells Expression of polκ by of a caused an of polκ. However, at this is not to extent this might the the extent of bypass was to the bypass in PolK+/+ cells. In polκ a major role in the bypass of the BP-G adduct and is for the bypass of at least two-thirds of the BP-G adducts in across the BP-G in the of the specificity of BP-G in of BP-G adducts was more than the of other in both PolK+/+ and PolK–/– cells. However, of the was in PolK–/– than in PolK+/+ cells plasmid of the bypass in PolK+/+ cells in with in PolK–/– cells plasmid bypass was more mutagenic in PolK–/– cells with PolK+/+ both the mutagenic was misinsertion of the BP-G revealed that in the PolK–/– cells transfected with the of the bypass were in such cells with the of the bypass were mutagenic polκ in PolK–/– cells TLS across the BP-G adduct mutagenic than in cells lacking specificity a BP-G adduct TLS in PolK+/+ and MEFs in the of polκ from a plasmid plasmid in a Polκ for TLS across a in the specificity of TLS by polκ, with gapped plasmids a synthetic abasic site. across the abasic of ± and ± in PolK+/+ and PolK–/– Analysis of the specificity of the abasic revealed that primarily were the abasic with results in human cells in S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). of were in PolK+/+ and PolK–/– cells These results indicate that polκ is not required for in vivo bypass across an abasic and specificity of bypass across an abasic in and coli number of in a with of ± ± The number of in a with of in a for across the BP-G the polymerase specificity for TLS, the ability of polη by the human gene to bypass BP-G lesions in in bypass in the human cell was ± and ± for plasmids and bypass in the human cell was ± and ± for plasmids and We also the specificity of the BP-G adduct and that in both cell bypass was in cells and in cells. This accuracy of bypass is higher than in the PolK+/+ MEFs The for the in accuracy is not from human and cells cell embryo cells. In polη is not required for bypass across the BP-G adduct in and specificity of bypass across BP-G adducts in human and number of in a with of ± ± ± ± The number of in a with of in a In across the BP-G by DNA the that the role of polκ in TLS across the BP-G adduct from the of other DNA polymerases to bypass this lesion. was that polη can bypass BP-G adducts F. X. O. Wang Z. 2000; 28: PubMed Google Scholar, D. H. C. Hanaoka F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, O. D. Wang Z. S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, TLS across BP-G is by DNA sequence M. S. A. S. T. 1996; PubMed Scopus Google Scholar, R. T. PubMed Scopus Google Scholar, T. A. PubMed Scopus Google Scholar, PubMed Scopus Google the ability of human polκ, polη, and to bypass the BP-G adduct in the DNA sequence that was used in in vivo the of TLS across the BP-G lesion by human DNA polymerases and was using DNA sequence is the as in the lesion plasmids used in the in vivo Polκ bypassed the BP-G adduct in of the sequence with high in In contrast, polη bypass at the sequence bypass was than by polκ. However, polη and polκ showed bypass the was was to bypass the adducts with results F. X. O. Wang Z. 2000; 28: PubMed Google Scholar, D. H. C. Hanaoka F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, O. D. Wang Z. S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google at least other DNA polη, is of a BP-G adduct in is not for this bypass in The of DNA from the Y that lesion bypass is an in mammalian cells from The of the of TLS polymerases and of in the cell quantitative We used a quantitative TLS in to the of polκ in TLS in cultured cells. The major of this are its specificity for TLS and its quantitative which are for the in of in vivo The of this are that is plasmid and that a than a the can be as a for DNA the is to the of DNA damage and to the of DNA as in this and in S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, S. Livneh Z. 2002; PubMed Scopus Google Scholar). The specificity of this assay for TLS is by the of the plasmid a repair is not to DNA (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995Google and in the of an recombinational repair as demonstrated S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the other can interfere with the DNA DNA which this can to the of abasic (1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995Google Scholar). This that a DNA were to the BP-G adduct, the due to abasic than the BP-G This is by was in bypass across an abasic in PolK+/+ and PolK–/– were in the bypass across BP-G in cell as is DNA that BP-G. the results with the gapped plasmid the BP-G adduct in the DNA can be to TLS in the mammalian cells. results indicate that the BP-G adduct is bypassed in mammalian cells with to This is more than higher than the extent of bypass in coli across the BP-G using the gapped plasmid These with results higher bypass across an abasic in mammalian cells with coli S. S. G. Livneh Z. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google indicate that TLS in is more efficient in mammalian cells than in coli and is therefore to be a more significant repair in than in coli. of polκ in to PolK–/– mouse embryo fibroblasts were to be to and by T. H. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google and the gene is subject to T. J. M. H. H. Genes 2001; PubMed Scopus Google Scholar). These results indicate that polκ an role in mouse cells from the and mutagenic of BP, with a role of polκ in TLS in However, the results can be by the of polκ in to DNA damage other than TLS, such as error-free repair and a polκ and the and and was in M. Wang Genes Dev. PubMed Scopus Google Scholar). was to be a T.A. Genes Dev. 1996; 10: PubMed Scopus Google Scholar). The in this for the of polκ in TLS across BP-G adducts in cells lacking polκ a in TLS across this and of human polκ in cells in of the bypass polκ is for the bypass of at least two-thirds of the BP-G In in the of polκ polymerase bypass BP-G to is that in PolK+/+ cells polκ is for the bypass of more than two-thirds of the BP-G This is the of DNA polymerases present in mammalian cells least DNA S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar, U. Trends Biochem. Sci. 2000; 25: Full Text Full Text PDF PubMed Scopus Google the of DNA damage specificity in the bypass of at least TLS DNA polymerases. The role of polκ in bypass across the BP-G adduct not from an of other DNA polymerases to bypass this polη was to bypass the BP-G adduct in of the with an efficiency to that of polκ. This is with the that regulation of TLS DNA polymerases an role in the TLS is that polκ is not the DNA polymerase that can bypass BP-G in bypass of were in cells lacking polκ. this the of polymerases is a is polη, its ability to bypass the BP-G adduct in as in this and in F. X. O. Wang Z. 2000; 28: PubMed Google Scholar, D. H. C. Hanaoka F. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, O. D. Wang Z. S. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is polκ in the bypass of DNA lesions other than BP-G. However, is that adducts with other be bypassed by polκ. was that polκ is a of in vitro R.E. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, L. Prakash L. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). this not polκ both the misinsertion and in vivo an polymerase is in the bypass Analysis of the specificity of the BP-G that the major mutagenic the misinsertion of with results Maher Mol. Cell. Biol. PubMed Scopus Google Scholar, A. T. S. M. PubMed Scopus Google Scholar). bypass was more mutagenic in the of polκ, in an higher of of This is not a to a the mutagenic of adducts in the of DNA lesions with the number of the results with indicate that polκ bypasses BP-G adducts with higher efficiency and higher accuracy than other DNA polymerases. This the of polη polκ the demonstrated of a TLS polymerase with the ability to bypass a lesion with higher accuracy than other DNA polymerases. results that at least DNA polymerases to bypass DNA lesions with higher efficiency and higher accuracy than In this polη and polκ DNA damage tolerance which are to the of unrepaired lesions in DNA by restoring DNA replication in the of with a of with other polymerases.