UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

Woodgate, Roger; Rajagopalan, Malini; Lu, Chi Zen; Echols, Harrison

doi:10.1073/pnas.86.19.7301

Cited by 203 publications

(191 citation statements)

References 42 publications

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“…Plasmid pRW66 produces roughly fourfold more UmuD' than that expressed from the chromosome (data not shown) and would therefore be expected to give rise to -10,000 UmuD' molecules per cell. Given that UmuD(D') is a dimer under physiological conditions (6,57), this equates to a ratio of approximately three UmuD' dimers to one UmuC molecule. Previously, we have noted that when expressed from the natural umuDC operon, UmuD(D') is produced in an approximately sixfold excess over UmuC (56).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremel useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

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Section: Resultsmentioning

confidence: 99%

Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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“…Gel filtration of UmuDЈ 2 or UmuD 2 shows that their elution volume is between the expected size of a dimer and that of a trimer (SI Fig. 10A) (5). Extensive evidence suggests dimeric forms of UmuD 2 , UmuDЈ 2 , and UmuDЈD (5), whereas no evidence for trimers has been found.…”

Section: Crowding Agents and Specific Protein-protein Interactions Inmentioning

confidence: 99%

“…Only UmuD 2 prevents DinB-induced -1 frame shifts (7), whereas only UmuDЈ 2 activates UmuC for TLS (4)(5)(6). UmuD 2 interacts preferentially with the ␤-processivity subunit of Pol III, whereas UmuDЈ 2 favors the ␣-catalytic subunit (8).…”

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confidence: 99%

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Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon

Sousa²,

Mohana‐Borges³

et al. 2008

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

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Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD 2 is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the Nterminal 24 amino acids of each UmuD. The remaining fragment, UmuD 2, is required for mutagenic TLS. The small proteins UmuD2 and UmuD 2 make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD 2 and UmuD 2 have circular dichroism (CD) spectra with almost no ␣-helix or ␤-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected ␤-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD 2 and UmuD 2 show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.natively unfolded ͉ SOS response ͉ unstructured ͉ denatured ͉ DNA repair

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“…The UmuDЈ 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 UmuDЈ 2 C complex is able to participate in TLS (43). In addition to participating in TLS, UmuC together with the full-length UmuD 2 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).…”

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confidence: 99%

Replicative DNA Polymerase

Encyclopedia of Cancer

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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 proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of 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 ␤ 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 ␤ 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 ␤ 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 ␤ are a subset of those between UmuD and ␤. Taken together, these findings suggest that interaction of ␤ 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 and the ␤ subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

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UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

Cited by 203 publications

References 42 publications

Isolation and characterization of novel plasmid-encoded umuC mutants

Isolation and characterization of novel plasmid-encoded umuC mutants

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Replicative DNA Polymerase

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