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

Sutton, Mark D.; Farrow, Mary F.; Burton, Briana M.; Walker, Graham C.

doi:10.1128/jb.183.9.2897-2909.2001

Cited by 41 publications

(64 citation statements)

References 67 publications

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“…pJD100 is a pWSK29 (54) derivative that contains the wild-type dnaN gene in an EcoRI-SalI fragment positioned downstream of the lac promoter. It was cloned in two steps using the EcoRI-XhoI fragment from pACYCdnaA (43) and the XhoI-SalI fragment from pJRC210 (42). In the absence of added isopropyl-␤-D-thiogalactopyranoside (IPTG), pJD100 expresses ␤ from the native dnaN promoters.…”

Section: Methodsmentioning

confidence: 99%

TheEscherichia coli dnaN159Mutant Displays Altered DNA Polymerase Usage and Chronic SOS Induction

Sutton

2004

J Bacteriol

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117

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The Escherichia coli ␤ sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the ␤ clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant ␤ clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant ␤ clamp protein (␤159) is impaired for interactions with the ␣ catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the ␤ clamp might help to coordinate protein traffic at the replication fork.It has become increasingly apparent in recent years that DNA replication, recombination, and repair are intimately related to each other (reviewed in reference 7). For example, the Escherichia coli RecA protein is not only required for most homologous recombination but is also required for DNA replication under normal growth conditions (7, 28), as well as for the proper regulation of global cellular responses following DNA damage (13, 47), including replication of damaged DNA (13,47). Moreover, the fact that the same DNA structure may serve as a substrate for both DNA replication and homologous recombination indicates that mechanisms must exist for coordinately regulating these processes in vivo (48). Consistent with the need for such regulation, processing of replication intermediates by recombination is proposed to serve as one cause of genome instability and is suspected to be a contributing factor to the development of certain cancers in humans (25). The fact that E. coli possesses at least five distinct DNA polymerases and that humans possess at least 16 DNA polymerases (reviewed in references 11, 12, and 48) further underscores the need organisms have for a higher-order regulatory system that acts to coordinate the actions of the cell's different DNA polymerases with those of its various repair and recombination pr...

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Section: Methodsmentioning

confidence: 99%

TheEscherichia coli dnaN159Mutant Displays Altered DNA Polymerase Usage and Chronic SOS Induction

Sutton

2004

J Bacteriol

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117

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“…On the basis of these findings, we have proposed that interaction of UmuDЈ with ␣ is important for TLS, perhaps as part of a polymerase-switching mechanism (4,25,(27)(28)(29), whereas interaction of UmuD with ␤ is important for enabling the UmuD 2 Cdependent checkpoint control (4,25,28,29). Although genetic and biochemical analyses are consistent with the UmuDЈ-␣ interaction being important for TLS in vivo (reviewed in refs.…”

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confidence: 99%

“…Viewed in this way, posttranslational modification of UmuD to yield UmuDЈ acts as a molecular switch to regulate temporally the two physiological roles of the umuDC gene products. In addition, specific interactions of UmuD and UmuDЈ with the ␣ (catalytic), (proofreading), and ␤ (processivity clamp) subunits of the E. coli replicative DNA polymerase, DNA polymerase III (pol III), are believed to be important for regulating which role the umuDC gene products will play (25)(26)(27)(28).…”

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confidence: 99%

Posttranslational modification of the umuD -encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the β processivity clamp

Sutton

Narumi²,

Walker³

2002

Proc. Natl. Acad. Sci. U.S.A.

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The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis. Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD proteins, with components of the replicative DNA polymerase (pol III), are important for determining which biological role the umuDC gene products will play. Here we report our biochemical characterizations of the interactions of UmuD and UmuD with the pol III ␤ processivity clamp. These analyses demonstrate that UmuD possesses a higher affinity for ␤ than does UmuD because of the N-terminal arm of UmuD (residues 1-39), much of which is missing in UmuD . Furthermore, we have identified specific amino acid residues of UmuD that crosslink to ␤ with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction. We have recently proposed a model for the solution structure of UmuD2 in which the N-terminal arm of each protomer makes extensive contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface. Taken together, our findings suggest that UmuD2 has a higher affinity for the ␤-clamp than does UmuD 2 because of the structures of its N-terminal arms. Viewed in this way, posttranslational modification of UmuD, which entails the removal of its N-terminal 24 residues to yield UmuD , acts in part to attenuate the affinity of the umuD gene product for the ␤-clamp. Implications of these structure-function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.

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“…The presence of the ␤ clamp is also thought to play a key role in managing polymerases at the replication fork (15,17). UmuD and UmuDЈ interact with the ␤ clamp physically, as well as genetically (5,42,46,48,49). Deletion of the N-terminal 9 residues of UmuD has little effect on its ability to crosslink to the ␤ clamp, while deletion of the N-terminal 19 residues results in a dramatic decrease in, but not a complete loss of, cross-linking efficiency (48).…”

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Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

et al. 2009

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Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD, comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD 2 C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuDC, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuDC-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

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

Cited by 41 publications

References 67 publications

TheEscherichia coli dnaN159Mutant Displays Altered DNA Polymerase Usage and Chronic SOS Induction

TheEscherichia coli dnaN159Mutant Displays Altered DNA Polymerase Usage and Chronic SOS Induction

Posttranslational modification of the umuD -encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the β processivity clamp

Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

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