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Dual Role for Mammalian DNA Polymerase Ζ in Maintaining Dual role for mammalian DNA polymerase ζ in PNAS PLUS maintaining genome stability and proliferative responses Sabine S. Langea, Ella Bedforda, Shelley Reha, John P. Wittschiebenb, Steve Carbajalc, Donna F. Kusewitta,d, John DiGiovannic, and Richard D. Wooda,d,1 aDepartment of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center Science Park, Smithville, TX 78957; bNovo Nordisk Foundation, 2900 Hellerup, Denmark; cDell Pediatric Research Institute, The University of Texas, Austin, TX 78723; and dGraduate School of Biomedical Sciences at Houston, Smithville, TX 78957 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved January 11, 2013 (received for review October 5, 2012) DNA polymerase ζ (polζ) is critical for bypass of DNA damage and the including ribonucleotides accidentally incorporated during DNA associated mutagenesis, but also has unique functions in mammals. synthesis (8). It is required for embryonic development and for viability of hema- S. cerevisiae can survive without polζ, but the enzyme clearly has topoietic cells, but, paradoxically, skin epithelia appear to survive unique functions in mammals because Rev3L-knockout mouse polζ deletion. We wished to determine whether polζ functions in embryos die in midgestation (9–11). To circumvent this difficulty a tissue-specific manner and how polζ status influences skin tumor- and to investigate the function of polζ in vivo, mice containing igenesis. Mice were produced in which Rev3L (the catalytic subunit loxP recombination sites flanking essential Rev3L exons have been of polζ) was deleted in tissues expressing keratin 5. Efficient epider- used for conditional disruption of polζ function. In a previous study mal deletion of Rev3L was tolerated but led to skin and hair abnor- we used Cre recombinase controlled by the mouse mammary tu- malities, accompanied by evidence of DNA breaks. Unchallenged mor virus (MMTV) promoter, which expresses in a mosaic fashion mice developed tumors in keratin 5-expressing tissues with age, in hematopoietic and epithelial cells. Normal hematopoietic cells consistent with the chromosomal instability accompanying a polζ did not survive MMTV-Cre–mediated Rev3L disruption, even in Rev3L defect. Unexpectedly, mice with the deletion were much more a p53-defective background (12). Similarly, inactivation of Rev3L sensitive to UVB radiation than mice defective in other DNA repair by CD21-Cre or CD19-Cre caused a loss of mouse B-cell viability ζ genes. Following irradiation, pol -defective mice failed to mount (13, 14). On the other hand, a mosaic of viable Rev3L-deleted and skin-regenerative responses and responded to stress by mobilizing nondeleted cells was present in skin epithelia of MMTV-Cre mice. melanocytes to the epidermis. However, they did not develop skin Cells that had deleted Rev3L were present in adult ear, skin, sali- tumors after chronic UVB irradiation. To determine the proliferative vary gland, and mammary gland (12). Therefore, deletion of Rev3L potential of polζ-deficient skin epithelia, keratinocytes were iso- in epidermally derived mammalian cells unexpectedly appeared lated and examined. These keratinocytes harbored chromosomal compatible with viability. gaps and breaks and exhibited a striking proliferation defect. These These observations raise several important questions. First, are results can be unified by a model in which slowly dividing cells ac- the requirements for polζ tissue specific, so that epidermal cells cumulate replication-associated DNA breaks but otherwise survive have a requirement for polζ that is fundamentally different from Rev3L deletion, but functional polζ is essential for responses requir- that of hematopoietic cells? Second, what is the importance of ing rapid proliferation, both in cell culture and in vivo. The results ζ reveal a biological role for mammalian polζ in tolerating DNA mammalian pol in defense against UV radiation in vivo? In- damage and enabling proliferative responses in vivo. vestigation of radiation sensitivity in mammalian cells has been limited previously to immortalized cells in culture. Third, if polζ DNA replication | double-strand breaks | UV radiation | carcinogenesis Significance ast, efficient genomic duplication requires that DNA be com- Fpletely intact and in the B form, because replicative DNA In mammalian cells DNA polymerase ζ (polζ) appears critical for polymerases cannot synthesize using a damaged DNA template bypass of DNA damage and was expected to be important for (1, 2). When such a template is encountered, replication halts, UV-induced skin carcinogenesis. To investigate the response to ζ and either a double-strand break (DSB) forms after replication UV radiation, we engineered mice lacking pol in the epidermis, fork collapse or the lesion is bypassed by translesion synthesis circumventing a requirement for embryonic development. These (TLS) polymerases or by template switching. After such damage mice were much more sensitive to UVB radiation than predicted, tolerance,theDNAcanberepaired.Therelativeimportanceof failed to mount skin-regenerative responses, and did not de- ζ fi each pathway for maintaining genomic stability and preventing velop UV-induced skin tumors. Even unirradiated pol -de cient keratinocytes had a marked proliferation defect and increased carcinogenesis is unknown. ζ TLS is mediated by specialized DNA polymerases (reviewed in chromosomal breaks. Thus in rapidly proliferating cells, pol ref. 3). DNA polymerase ζ (polζ, catalytic subunit REV3L) stands maintains levels of DNA breaks below a lethal threshold. out as the most important DNA polymerase for bypass of lesions in GENETICS ζ Author contributions: S.S.L., E.B., J.P.W., S.C., D.F.K., J.D., and R.D.W. designed research; template DNA. Pol plays a major role in the bypass of many types S.S.L., E.B., S.R., and S.C. performed research; S.C. and J.D. contributed new reagents/ of DNA damage, including pyrimidine(6-4)pyrimidone photo- analytic tools; S.S.L., E.B., S.R., J.P.W., D.F.K., and R.D.W. analyzed data; and S.S.L., J.P.W., products induced by UV radiation (4, 5) as well as lesions formed by and R.D.W. wrote the paper. chemical damaging agents such as cisplatin and benzo[a]pyrene (4). The authors declare no conflict of interest. Polζ also can be used for bypass of the frequent endogenously This article is a PNAS Direct Submission. formed abasic sites in DNA (4). In the yeast Saccharomyces cer- Freely available online through the PNAS open access option. evisiae, spontaneous and DNA damage-induced mutagenesis is 1To whom correspondence should be addressed. E-mail: [email protected]. ζ highly dependent on pol function (6), and the enzyme is involved This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the bypass of common impediments to DNA replication (7), 1073/pnas.1217425110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1217425110 PNAS | Published online February 5, 2013 | E687–E696 Downloaded by guest on October 2, 2021 is important for bypass of UV radiation damage, how does it Results modulate skin carcinogenesis in mammals? Mice Survive Epithelial Deletion of Rev3L but Have Skin Tissue Ab- Humans and mice defective in the DNA nucleotide excision normalities. Mice containing a floxed allele of Rev3L (12, 15) cou- repair (NER) pathway (NER) or in DNA polymerase η (polη) pled with either a wild-type allele or a knockout Rev3L allele (Fig. have a greatly elevated predisposition to UV radiation-induced skin 1A) were bred with transgenic mice expressing Cre recombinase cancer. A major contributor to thistumorigenesisistheaccumu- from a keratin 5 promoter (BK5.Cre mice). This promoter drives lation of point mutations in key driver genes. Polζ-depleted cells in gene deletion at high efficiency (≥95%) in the basal epithelia culture are reported to have reduced UV-induced point mutations of skin epidermis, hair follicles, sebaceous glands, thymus, and (5, 6). It is possible that suppression of UV-induced mutagenesis mammary gland (17, 18), but not in hematopoietic tissues. The in key genes for skin carcinomas would diminish tumorigenesis in efficiency of Cre activity was monitored using the membrane- polζ-deleted epidermis. On the other hand, the observed increase Tomato/membrane-Green (mT/mG) Cre reporter gene (15, 19) in the frequency of chromosome rearrangements in Rev3L-deleted (Fig. 1B) and by PCR of genomic DNA (Fig. S1). Control BK5. cells (13, 15, 16) might accelerate cancer development. Mice effi- Cre;Rev3L+/lox and BK5.Cre;Rev3L+/+ mice retained one or two ciently deleting Rev3L in keratin 5-expressing epithelia were used functional alleles of Rev3L, respectively, while still expressing in this study, allowing us to ask definitively whether normal epi- keratin 5-driven Cre recombinase. − thelial tissues can develop and renew normally in vivo in the ab- Mice deleting polζ in basal epithelium (BK5.Cre;Rev3L /lox) sence of polζ.Wefind a major biological role of polζ in the defense were viable but were underrepresented at weaning (Fig. 1C). In against UV radiation-induced DNA damage in vivo. The results addition, they displayed defects in skin and hair growth. The BK5. − show that mammalian polζ is important not only for tolerating Cre;Rev3L /lox pups were recognizable by a thinner hair coat (Fig. DNA lesions but also for allowing cells to proliferate after damage. 1D), with hair becoming more normal in appearance by 8 wk of AB C DE Fig. 1. Abnormal skin epithelia and increased morbidity in aging Rev3L mice. (A) Schematic of (i) wild-type, (ii) knockout, (iii) floxed-intact, and (iv) floxed-deleted alleles of the murine Rev3L gene (12, 15). Vertical bars represent exons (red bars contain part of polymerase motif I and all of motif V); tri- angles are loxP sites. (B) mT/mG skin and salivary gland expressing RFP (no Cre activity) or GFP (Cre activity) in BK5.Cre transgenic mice. (C) Genotype percentages from a cross of male BK5.Cre;Rev3L+/lox − FG and female Rev3L+/ mice.
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