Replicative DNA Polymerase

Abstract

The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD 2 C. We have previously reported that overexpression of the proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of with UmuD 2 C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the ␤ processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant ␤ proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of ␤ or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD and ␤ are a subset of those between UmuD and ␤. Taken together, these findings suggest that interaction of ␤ with UmuD 2 C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD 2 C with the and the ␤ subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.