Two processivity clamp interactions differentially alter the dual activities of UmuC

Abstract

SummaryDNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical β β β β processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type β β β β can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and β β β β -dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in β β β β , we restored UV-induced mutagenesis by a UmuC β β β β -binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with β β β β suggests that, in addition to the canonical β β β β -binding motif, a second site of pol IV ( 303 VWP 305 ) interacts with β β β β at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the coldsensitive phenotype. These two β β β β interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the β β β β clamp.

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

“…A putative interaction with the rim site could also position the other E. coli Y-family polymerase, Pol V, on the clamp when it is expressed later in the SOS damage response (37). This binding activity would create a hierarchy for access to the primer terminus, a view of the toolbelt model in which clamp-polymerase interactions do more than merely increase the local polymerase concentration at the DNA template.…”

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

“…The roles of the E. coli ␤ clamp in translesion synthesis are well established (5,8,30,31). Binding sites on the E. coli ␤ clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD 2 ЈC) have been identified, and the consequence of disrupting their association with the ␤ clamp has illustrated the critical importance of the ␤ clamp to the activity of both of these polymerases (4,5,8,26,30,31,48,49).…”

“…The roles of the E. coli ␤ clamp in translesion synthesis are well established (5,8,30,31). Binding sites on the E. coli ␤ clamp that accommodate translesion polymerases pol IV (DinB) and pol V (UmuD 2 ЈC) have been identified, and the consequence of disrupting their association with the ␤ clamp has illustrated the critical importance of the ␤ clamp to the activity of both of these polymerases (4,5,8,26,30,31,48,49).In addition to the involvement of the ␤ clamp in replication initiation, DNA replication, and translesion synthesis, the E. coli and B. subtilis ␤ clamp also functions in DNA mismatch repair (MMR) (45,46,64). The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60).…”

Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli

“…Fifteen micrograms of total protein of each lysate was loaded onto a 4 to 20% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride membrane and probed with an anti-DinB antibody, under Western conditions described previously (2) except with the addition of a high-salt (0.5 M NaCl) wash after incubation with the secondary antibody. Antibodies against UmuD and UmuC can detect plasmid-borne protein levels only after SOS induction.…”

“…Thus, the expression of umuDC under the conditions used for nusA11 multicopy suppression cannot be detected by Western blotting. In order to check that the different mutants used were stably expressed compared to the wild type, AB1157 cells harboring plasmids described above (C) were irradiated with UV to induce the SOS response, and Western blot assays were then performed as described previously (2 (18,20,23,39). Alternatively, gaps opposite lesions could also be formed by UvrABC-dependent nucleotide excision repair if two lesions are very close together but on opposing strands or by UvrABC-dependent incisions during the repair of an intrastrand cross-link (12).…”