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

Abstract

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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