The mutagenesis protein MucB interacts with single strand DNA binding protein and induces a major conformational change in its complex with single-stranded DNA.
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 SSB.ssDNA 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 SOS-regulated mutagenesis.
Sarov-Blat
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L
Department of Biological Chemistry, Faculty of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Livneh
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Z
D001426:Bacterial Proteins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry; D004996:Ethidium / Q000378:metabolism; D016296:Mutagenesis / Q000235:genetics; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism; D013014:SOS Response, Genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C063757:MucA protein, Bacteria; C093590:MucB protein, Bacteria; D011938:Rec A Recombinases; D004996:Ethidium