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

Beuning, Penny J.; Chan, Sarah; Waters, Lauren S.; Addepalli, Haripriya; Ollivierre, Jaylene N.; Walker, Graham C.

doi:10.1128/jb.00292-09

Cited by 15 publications

(22 citation statements)

References 52 publications

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“…We observed that strains expressing UmuC S31A had a growth defect and subsequently identified populations of strains expressing UmuC S31A with severe, intermediate, or no growth defects, indicating that this variant acquired suppressor mutations. Strains expressing UmuC S31L did not have this growth defect (5). The location and behavior of S31A as well as those of S31L suggest that this residue plays an important role in UmuC function and perhaps, more specifically, in template DNA alignment in the active site.…”

Section: Discussionmentioning

confidence: 88%

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011

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DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD, form the specialized E. coli Y-family DNA polymerase UmuD 2 C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.Escherichia coli has five DNA polymerases that replicate DNA under different circumstances (22). The replicative polymerase in E. coli is DNA polymerase III (Pol III), a member of the C family. DNA polymerases IV and V are members of the Y family, which specialize in copying damaged DNA in a process known as translesion synthesis (TLS) (22, 47). Y-family DNA polymerases also copy undamaged DNA in an errorprone manner, possibly subjecting DNA to untargeted mutagenesis and potentially leading to antibiotic resistance or cancer (16,17,22,52).E. coli DNA polymerases IV (DinB) and V (UmuDЈ 2 C) are the products of the dinB and umuDC genes, respectively. Due to their potentially mutagenic nature, these proteins are highly regulated in a specific cellular response to DNA damage and other stresses called the SOS response (22,55). The SOS response is initiated when single-stranded DNA (ssDNA) forms downstream from a lesion in DNA due to the inability of the replicative DNA polymerase to copy damaged DNA. RecA then coats the ssDNA to form a RecA-ssDNA nucleoprotein filament, which is the inducing signal for the SOS response. The RecA-ssDNA filament facilitates the autocleavage of LexA, the repressor of the SOS genes, allowing the expression of at least 57 genes (22,66). While many genes are induced in the SOS response, expression of only the umuDC genes is required for SOS mutagenesis (71).The umuDC genes in E. coli encode UmuD, a polymerase manager protein, and UmuC, t...

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Section: Discussionmentioning

confidence: 88%

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011

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“…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).…”

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confidence: 99%

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

et al. 2010

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The ␤ clamp is an essential replication sliding clamp required for processive DNA synthesis. The ␤ clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of ␤ clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the ␤ clamp, a common site occupied by proteins that bind the ␤ clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis ␤ clamp separate the role of the ␤ clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24,(39)(40)(41). Encoded by the dnaN gene, the ␤ clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The ␤ clamp is a head-to-tail, ringshaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the ␤ clamp is proliferating cell nuclear antigen (PCNA) (15,28,40,41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40,41). Although the primary structures of the ␤ clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the ␤ clamp is not limited to its well-defined role in genome replication. The Escherichia coli ␤ clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19,37,43). This allows the ␤ clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the ␤ clamp binds YabA, a negative regulator of DNA replication initiation (12,29,52). It has also been suggested that the B. subt...

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“…The operator sequences of pGY9739 and pGY9738 contain the o 1 c mutation where a single base substitution leads to modestly increased expression of umuD and umuC (28,29). Strains were grown in Luria broth at 37°C supplemented with spectinomycin (60 g/ml) or ampicillin (100 g/ml).…”

Section: Methodsmentioning

confidence: 99%

“…Expression of UmuD and UmuDЈ proteins were accomplished as previously described (31). Cells were harvested, and UmuD and UmuDЈ proteins were purified according to published methods (28). The ␤ clamp was also purified using the method published for UmuD and UmuDЈ.…”

Section: Methodsmentioning

confidence: 99%

The Dimeric SOS Mutagenesis Protein UmuD Is Active as a Monomer

Ollivierre¹,

Sikora²,

Beuning

2011

Journal of Biological Chemistry

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The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD 2 is composed of 139-amino acid subunits and is up-regulated as part of the SOS response. Subsequently, damage-induced RecA⅐ssDNA nucleoprotein filaments mediate the slow self-cleavage of the N-terminal 24-amino acid arms yielding UmuD 2 . UmuD 2 and UmuD 2 make a number of distinct protein-protein contacts that both prevent and facilitate mutagenic translesion synthesis. Wild-type UmuD 2 and UmuD 2 form exceptionally tight dimers in solution; however, we show that the single amino acid change N41D generates stable, active UmuD and UmuD monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB) and the ␤ clamp. Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.

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

Cited by 15 publications

References 52 publications

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

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

The Dimeric SOS Mutagenesis Protein UmuD Is Active as a Monomer

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