Genetic Interactions between the Escherichia coli umuDC Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase

The umuDC genes encode a DNA polymerase, DNA polymerase V (Pol V), that has a remarkable ability to copy over abasic sites (43, 63), cyclobutane dimers (62), and pyrimidine-pyrimidone [6-4] photoproducts (62), a process referred to as translesion DNA synthesis (TLS). This ability, however, comes at the cost of reduced fidelity. Thus, replication by Pol V is inherently less accurate, leading to the formation of mutations, even when replicating undamaged templates (29, 62). Therefore, to limit the ability of Pol V to introduce mutations into the host genome, expression of the umuDC genes is tightly regulated as part of the Escherichia coli stress-induced SOS response (12). This highly regulated response helps the cell maintain the integrity of its genome following treatments that lead either directly or indirectly to DNA damage (12). The E. coli SOS response consists of at least 30 unlinked genes (9, 12), collectively referred to as the SOS regulon. Expression of the various SOS-regulated genes is coordinately regulated at the level of their transcription by the LexA and RecA proteins (28). In the absence of DNA damage, LexA acts to repress the expression of the members of the SOS regulon (27). RecA protein, the main bacterial recombinase required for essentially all homologous recombination (reviewed in reference 22), binds to single-stranded DNA (ssDNA) generated by the cell's failed attempts to replicate past lesions in its genome, thus forming RecA-ssDNA nucleoprotein filaments (47). These RecA-ssDNA filaments, in addition to acting in homologous recombination, also act to facilitate the latent capacity of LexA to autodigest (26). Autodigestion of LexA serves to inactivate it as a transcriptional repressor, leading to the concomitant increase in expression of LexA-regulated genes (12). The UmuD protein similarly undergoes a RecA-ssDNA-facilitated autodigestion that serves to remove its first 24 residues to yield UmuD′ (3, 34, 48). The UmuD′2 homodimer then interacts with UmuC (18, 59, 68), which has an ability to catalyze the formation of phosphodiester bonds, in such a way that the UmuD′2C complex is able to participate in TLS (43). In addition to participating in TLS, UmuC together with the full-length UmuD2 homodimer participates in a DNA damage checkpoint control that acts to regulate DNA replication in response to DNA damage, thereby allowing additional time for nucleotide excision repair to accurately remove lesions in the DNA prior to continued replication (37). Thus, self-cleavage of UmuD to UmuD′ can be regarded as a molecular switch that acts to temporally regulate these two distinct activities of the UmuD2C and UmuD′2C complexes (37, 57). In addition to participating in a DNA damage checkpoint control and enabling TLS, overexpression of the umuDC gene products confers a cold sensitivity for growth (30, 38, 59). Our recent characterizations of umuDC-mediated cold sensitivity indicated that (i) moderately elevated levels of the umuDC gene products confer a cold-sensitive growth phenotype, while similarly elevated levels of the umuD′C gene products do not (59), and (ii) the catalytic DNA polymerase activity of UmuC is not required for this cold sensitivity (59). These findings, together with others, suggest that the cold sensitivity conferred by elevated levels of the umuDC gene products is a manifestation of the inappropriate expression of UmuD2C functions involved in the DNA damage checkpoint control (37, 38, 59). Therefore, in an effort to better characterize the components of the UmuD2C-dependent checkpoint control, we have embarked on an analysis of the genetic requirements of umuDC-mediated cold sensitivity. We have previously suggested that interactions of the umuDC gene products with components of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme (Pol III), could serve as a convenient mechanism for regulating the checkpoint and TLS roles of the umuDC gene products (37, 57). On the basis of this hypothesis, we reasoned that if interactions involving the umuDC gene products and components of Pol III were important for the checkpoint role of UmuD2C, we might then be able to observe an effect on the extent of the cold sensitivity conferred by umuDC by the simultaneous overexpression of certain (i.e., relevant) components of Pol III. Using such an approach, we have recently reported that overproduction of the ɛ proofreading subunit of Pol III or deletion of its structural gene (dnaQ) suppresses umuDC-mediated cold sensitivity (56). A systematic analysis of the remaining nine Pol III subunits indicated that the homodimeric β processivity clamp was the only other Pol III subunit that when overexpressed affected the extent of umuDC-mediated cold sensitivity (56). In this report, we describe how overexpression of the β processivity clamp (encoded by dnaN) strongly exacerbates umuDC-mediated cold sensitivity. We have exploited this ability of the β clamp to confer a cold-sensitive growth phenotype upon a umuD′C-expressing E. coli strain (a strain that is not normally cold sensitive [59]) to identify novel dnaN alleles unable to confer this phenotype. Our genetic characterizations of these novel dnaN alleles indicate that they are also unable to exacerbate the cold sensitivity conferred by elevated levels of the umuDC gene products. Our results described in this report, taken together with others (37, 38, 56, 57, 59), suggest that the UmuD2C-dependent DNA damage checkpoint control is a manifestation of protein-protein interactions involving UmuD2C and the ɛ and the β subunits of Pol III.