Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins

Jonczyk, Piotr; Nowicka, Andadrianna

doi:10.1128/jb.178.9.2580-2585.1996

Cited by 37 publications

(33 citation statements)

References 54 publications

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“…Similar results were observed for UmuD, consistent with our hypothesis that UmuD and UmuDЈ might make similar contacts with UmuC. Furthermore, our finding that UmuD and UmuDЈ retained limited abilities to interact with all four derivatives of MBP-UmuC is consistent with the finding by Jonczyk and Nowicka (14) that N-terminal sequences of UmuC may also important for its interaction with UmuDЈ.…”

Section: Vol 183 2001supporting

confidence: 82%

“…Although Jonczyk and Nowicka did not detect an interaction between UmuD and UmuC in vivo using the yeast two-hybrid system, they did detect an interaction between UmuDЈ and UmuC (14). Furthermore, they reported that small deletions of either N-or C-terminal sequences of UmuC destroyed its ability to interact with UmuDЈ as measured with the yeast two-hybrid system (14).…”

Section: Resultsmentioning

confidence: 99%

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umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control

Sutton

Walker

2001

J Bacteriol

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The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD 2 C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD 2 C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD 2 C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

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Section: Vol 183 2001supporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control

Sutton

Walker

2001

J Bacteriol

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“…47). Interestingly, it has been reported that UmuC interacts with UmuDЈ, but not with UmuD, in the yeast two-hybrid system (45). In our case, MucB was found to interact both with MucA and MucAЈ.…”

Section: Discussionsupporting

confidence: 49%

“…It was previously shown that the UmuDЈ and UmuD proteins form both homo-and heterodimers when assayed biochemically (35,36,45) and in the yeast two-hybrid system (45). Here we show that MucAЈ and MucA behave similarly and exhibit both self-interactions and an interaction with each other.…”

Section: Discussionmentioning

confidence: 50%

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

Sarov‐Blat

Livneh

1998

Journal of Biological Chemistry

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The MucA and MucB proteins are plasmid-encoded homologues of the Escherichia coli UmuD and UmuC proteins, respectively. These proteins are required for SOS mutagenesis, although their mechanism of action is unknown. By using the yeast two-hybrid system we have discovered that MucB interacts with SSB, the single strand DNA binding protein (SSB) of E. coli. To examine the interaction at the protein level, the MucA, MucA, and MucB proteins were overproduced, purified in denatured state, and refolded. Purified MucA and MucA each formed homodimers, whereas MucB was a monomer under native conditions. RecA promoted the cleavage of MucA to MucA, and MucB was found to bind single-stranded DNA (ssDNA), similarly to the properties of the homologous UmuD and UmuC proteins. Purified MucB caused a shift in the migration of SSB in a sucrose density gradient, consistent with an interaction between these proteins. Addition of MucB to SSB-coated ssDNA caused increased electrophoretic mobility of the nucleoprotein complex and increased staining of the DNA by ethidium bromide. Analysis of radiolabeled SSB in the complexes revealed that only a marginal release of SSB occurred upon addition of MucB. These results suggest that MucB induces a major conformational change in the SSB⅐ssDNA complex but does not promote massive release of SSB from the DNA. The interaction with SSB might be related to the role of MucB in SOSregulated mutagenesis.UV mutagenesis in Escherichia coli is a regulated process, controlled by the SOS stress response through its two global regulators, RecA and LexA. The mechanism underlying this process is trans-lesion replication by a DNA polymerase, most likely DNA polymerase III (for reviews see Refs. 1-3). This process requires specifically two SOS-induced proteins, UmuD and UmuC (4 -6), whose mechanism of action is unknown. A prevailing hypothesis is that UmuDЈ, the active form of UmuD (7-9), along with UmuC are required to assist the DNA polymerase in replicating the damaged site (10). However, the nature of this assistance is not clear because purified DNA polymerases can bypass DNA lesions unassisted (11)(12)(13)(14)(15)(16)(17)(18)(19). Moreover, in an in vitro system for UV mutagenesis carried out with crude protein extracts (20, 21) or with purified proteins (22), we have found that UV mutations were effectively produced in the absence of UmuDЈ and UmuC.Homologues of UmuD and UmuC have been identified in other bacteria, and some of them are encoded by conjugational plasmids (23, 24). The most well-studied of these are the mucA and mucB genes, encoding homologues of UmuD and UmuC, respectively (25,26). An approach that has proven useful in the study of many proteins is to examine their interactions with other proteins. Employing this approach we present here in vivo and in vitro data that show, for the first time, that MucB interacts with SSB 1 and greatly changes the structure of the SSB⅐ssDNA complex. EXPERIMENTAL PROCEDURESMaterials-The sources for the materials used in this study were as follows: isoprop...

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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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Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins

Cited by 37 publications

References 54 publications

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

Replicative DNA Polymerase

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Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins

Cited by 37 publications

References 54 publications

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD 2 C Complex Involved in a DNA Damage Checkpoint Control

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD 2 C Complex Involved in a DNA Damage Checkpoint Control

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

Replicative DNA Polymerase

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Resources

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umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control

umuDC -Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD ₂ C Complex Involved in a DNA Damage Checkpoint Control