DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients - PubMed (original) (raw)

DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Omer Ziv et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

TLS across a TT CPD in XPV cells pretreated with siRNA against specific TLS polymerases. (A) Outline of the gapped plasmid TLS assay. See text for details. (B) Relative TLS extent across TT CPD lesions in XPV cells pretreated with siRNA against TLS polymerases. Each column represents the average of 6–10 measurements. See details in

Table S1

and in Materials and Methods. (C) Extent of accurate and mutagenic TLS in XPV cells pretreated with siRNA against TLS polymerases. Sequences were classified as accurate TLS (insertion of AA opposite the TT CPD), mutagenic TLS (nucleotides other than AA inserted opposite the TT CPD, or mutations at the nucleotides flanking the TT CPD), and non-TLS events (large insertions and deletions). The extent of each event type was obtained by multiplying the extent of plasmid repair (

Table S1

) by the fraction of that event obtained from the DNA sequence analysis (

Table S2

), as presented in

Table S3

.

Fig. 2.

Fig. 2.

UV sensitivity of XPV and normal cells pretreated with siRNA against TLS polymerases. (A) Outline of the experimental scheme. (B and C) XPV cells (B) or normal MRC5 human cells (C) were transfected with siRNA, and UV irradiated after 48 h. For MRC5 cells, 1 mM caffeine was added immediately following UV irradiation. Viability was determined 48 h after UV irradiation by measuring cellular ATP as described in Material and Methods.

Fig. 3.

Fig. 3.

UV sensitivity of XPA cells pretreated with siRNA against TLS polymerases. Cells were transfected with siRNA, and UV irradiated after 48 h at the indicated doses. Viability was determined 48 h after UV irradiation by measuring cellular ATP (A) or 12 days after UV irradiation by measuring colony forming ability (B). (C) XPA cells stably expressing CPD photolyase were UV irradiated at 3 Jm−2 and immediately illuminated with visible light to photoreactivate CPD lesions. Cell viability was determined as in A. See Materials and Methods for details.

Fig. 4.

Fig. 4.

Model describing TLS across CPD in human cells. In human normal cells polη carries out efficient and relatively accurate TLS across CPD. In human XPV cells, TLS across CPD is performed by a back-up system in 2-polymerase reactions, in which polκ or polι perform insertion opposite the CPD, whereas polζ performs the extension step. This pathway is less efficient and more mutagenic than the polη-dependent pathway. A possible 3-polymerase mechanism is also presented. See text for details.

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References

    1. Friedberg EC, et al. DNA Repair and Mutagenesis. 2nd edition. Washington DC: ASM Press; 2006.
    1. Livneh Z. DNA damage control by novel DNA polymerases: Translesion replication and mutagenesis. J Biol Chem. 2001;276:25639–25642. - PubMed
    1. Prakash S, Johnson RE, Prakash L. Eukaryotic translesion synthesis DNA polymerases: Specificity of structure and function. Annu Rev Biochem. 2005;74:317–353. - PubMed
    1. Yang W, Woodgate R. What a difference a decade makes: Insights into translesion synthesis. Proc Natl Acad Sci USA. 2007;104:15591–15598. - PMC - PubMed
    1. Gan GN, Wittschieben JP, Wittschieben BO, Wood RD. DNA polymerase ζ (pol ζ) in higher eukaryotes. Cell Research. 2008;18:174–183. - PubMed

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