Replication-associated repair of adenine:8-oxoguanine mispairs by MYH.

Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG.

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice.

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.