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Mutagenesis Is Elevated in Male Germ Cells Obtained from DNA Polymerase-beta Heterozygous Mice¹

Departments of Cellular and Structural Biology⁴ and Pathology,⁵ and The Barshop Center for Longevity and Aging Studies,⁶ The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 Department of Pharmacology and Chemical Biology,⁷ University of Pittsburgh School of Medicine and The University of Pittsburgh Cancer Institute,⁸ Hillman Cancer Center, Pittsburgh, Pennsylvania 15213 The Laboratory of Structural Biology,⁹ National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 South Texas Veteran's Health Care System,¹⁰ Audie Murphy Memorial Veterans Affairs Hospital, San Antonio, Texas 78229

ABSTRACT

Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA polymerase-beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency..

base excision repair, DNA repair, gamete biology, gametogenesis, lacI, mutagenesis, POLB, spermatogenesis, spermatogenic cells

¹Supported by grant P20 CA103730 from the Elsa U. Pardee Foundation and the National Institutes of Health to R.W.S. and by grants 1 R01 AG24364-01 and AG21163 from the National Institutes of Health to C.A.W. This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. The contents are solely the responsibility of the authors and do not necessarily reflect the views of the funding agencies.

Correspondence: ²Christi A. Walter, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, Mail Code 7762, San Antonio, TX 78229-3900. FAX: 210 567 3803; e-mail: walter{at}uthscsa.edu

³Current address: Department of Molecular Embryology, Institute of Experimental Medicine, and Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic.