Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase

Citation
Md. Sutton et al., Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase, J BACT, 183(9), 2001, pp. 2897-2909
Citations number
68
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF BACTERIOLOGY
ISSN journal
0021-9193 → ACNP
Volume
183
Issue
9
Year of publication
2001
Pages
2897 - 2909
Database
ISI
SICI code
0021-9193(200105)183:9<2897:GIBTEC>2.0.ZU;2-Z
Abstract
The Escherichia coli umuDC gene products encode DNA polymerase V, which par ticipates in both translesion DNA synthesis (TLS) and a DNA damage checkpoi nt 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 enabl es TLS), In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inapprop riate expression of the DNA damage checkpoint control activity of UmuD(2)C. We have previously reported that overexpression of the epsilon proofreadin g subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon 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 beta processivity clamp of the E. coli r eplicative DNA polymerase (encoded by the dnaN gene) not only exacerbates t he 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 s train is not otherwise normally cold sensitive, To identify mutant beta pro teins possibly deficient for physical interactions with the umuDC gene prod ucts, 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 beta or prem aturely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN, Each of the dnaN missense mutations reta ined at least a partial ability to function in chromosomal DNA replication in vivo, In addition, these eight dnaN alleles were also unable to exacerba te the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta, Taken together, these findings sug gest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for h ow interactions of UmuD(2)C with the epsilon and the beta subunits of DNA p olymerase III might help to regulate DNA replication in response to DNA dam age are discussed.