engleski

Bacterial single strandend DNA binding protein - a novel target of tyrosine kinase

Single stranded DNA binding proteins (SSBs) are ubiquitous proteins that play an essential role in various stages of DNA metabolism. These proteins bind DNA in a sequence independent manner and maintain genome integrity during DNA replication, recombination or repair. Besides stabilizing single-stranded DNA (ssDNA), SSBs interact with enzymes such as DNA polymerase, RNA polymerase or DNA helicase and modulate their activities. Although accomplishing similar functions, bacterial and eukaryotic SSBs differ considerably in their structure. It is known that eukaryotic SSBs are regulated by phosphorylation on several serine and threonine residues. Until this study phosphorylation of SSB proteins in bacteria has not been recognized. By immunoaffinity chromatography we purified tyrosine phosphorylated SSB from Streptomyces griseus. Since genes encoding protein-tyrosine kinases have not been recognized in streptomycetes and SSBs from Streptomyces coelicolor and Bacillus subtilis share 40% identity, we used a B. subtilis protein-tyrosine kinase YwqD to examine phosphorylation of the two cognate SSBs (SSB and YwpH) in vitro. Focusing on this system we found that phosphorylation of SSB is affected antagonistically by B. subtilis tyrosine kinase (YwqD) and phosphatase (YwqE). Further, phosphorylation of B. subtilis SSB significantly increased binding to single-stranded DNA in vitro. We also found that under DNA-damaging conditions in B. subtilis cells SSB was phosphorylated to a significantly lower extent suggesting one biological aspect of this process in the regulation of DNA metabolism. Mass spectrometry analyzes showed that in vivo phosphorylation of B. subtilis SSB occurs on tyrosine residue 82 that is highly conserved in SSBs from Gram-positive bacteria. However, we also observed that tyrosine phosphorylation of B. subtilis, S. coelicolor, and Escherichia coli SSBs occured while they were expressed in E. coli. Our results indicate that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria.

SSB ; tyrosine phosphorylation ; bacteria

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