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Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli

Bharti, Sanjay Kumar and Rex, Kervin and Sreedhar, Pujari and Krishnan, Neeraja and Varshney, Umesh (2011) Chimeras of Escherichia coli and Mycobacterium tuberculosis Single-Stranded DNA Binding Proteins: Characterization and Function in Escherichia coli. In: PLoS One, 6 (12).

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Abstract

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m beta 1, m beta 1'beta 2, m beta 1-beta 5, m beta 1-beta 6 and m beta 4-beta 5) by transplanting beta 1, beta 1'beta 2, beta 1-beta 5, beta 1-beta 6 and beta 4-beta 5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m beta 1'beta 2(ESWR) SSB was generated by mutating the MtuSSB specific `PRIY' sequence in the beta 2 strand of m beta 1'beta 2 SSB to EcoSSB specific `ESWR' sequence. Biochemical characterization revealed that except for m beta 1 SSB, all chimeras and a control construct lacking the C-terminal domain (Delta C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m beta 1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m beta 1-beta 6, MtuSSB, m beta 1'beta 2 and m beta 1-beta 5 SSBs complemented E. coli Delta ssb in a dose dependent manner. Complementation by the m beta 1-beta 5 SSB was poor. In contrast, m beta 1'beta 2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

Item Type: Journal Article
Additional Information: This article is licensed under Creative Commons Attribution License.
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Depositing User: Id for Latest eprints
Date Deposited: 27 Jan 2012 06:31
Last Modified: 27 Jan 2012 06:39
URI: http://eprints.iisc.ac.in/id/eprint/43153

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