Sutton M D, Farrow M F, Burton B M, Walker G C
Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Bacteriol. 2001 May;183(9):2897-909. doi: 10.1128/JB.183.9.2897-2909.2001.
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 epsilon proofreading 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 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 beta 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 beta 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 beta are a subset of those between UmuD and beta. Taken together, these findings suggest 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 how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.
大肠杆菌umuDC基因产物编码DNA聚合酶V，其参与跨损伤DNA合成（TLS）和DNA损伤检查点控制。umuDC基因产物的这两个在时间上不同的作用是由RecA-单链DNA促进的UmuD（参与检查点控制）的自我切割来调节的，从而产生UmuD'（能够进行TLS）。此外，即使umuDC基因产物适度过表达也会导致冷敏感生长表型，这显然是由于UmuD(2)C的DNA损伤检查点控制活性的不适当表达。我们之前报道过，DNA聚合酶III的ε校对亚基的过表达会抑制umuDC介导的冷敏感性，这表明ε与UmuD(2)C的相互作用对于umuDC基因产物的DNA损伤检查点控制功能很重要。在此，我们报道大肠杆菌复制性DNA聚合酶的β持续性钳（由dnaN基因编码）的过表达不仅会加剧umuDC基因产物水平升高所带来的冷敏感性，而且还会使表达适度升高水平的umuD'C基因产物的菌株产生严重的冷敏感表型。这样的菌株在其他方面通常对冷不敏感。为了鉴定可能与umuDC基因产物缺乏物理相互作用的突变β蛋白，我们筛选了在过表达umuD'C的菌株上不能赋予冷敏感生长表型的新型dnaN等位基因。我们总共鉴定出75个dnaN等位基因，其中62个要么降低了β的表达，要么过早地截断了其合成，而其余等位基因定义了dnaN的8个独特错义突变。每个dnaN错义突变在体内染色体DNA复制中至少保留了部分功能能力。此外，这8个dnaN等位基因也不能加剧umuDC基因产物适度升高水平所带来的冷敏感性，这表明UmuD'与β之间的相互作用是UmuD与β之间相互作用的一个子集。综上所述，这些发现表明β与UmuD(2)C的相互作用对于umuDC基因产物的DNA损伤检查点功能很重要。我们讨论了UmuD(2)C与DNA聚合酶III的ε和β亚基之间的相互作用如何可能有助于响应DNA损伤来调节DNA复制的四种可能模型。
