Open AccessCloning, over‐expression and biochemical characterization of the single‐stranded DNA binding protein from Mycobacterium tuberculosis
Author(s) -
Purnapatre Kedar,
Varshney Umesh
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00684.x
Subject(s) - dna , biology , dna replication , protein–dna interaction , oligonucleotide , peptide sequence , amino acid , in vitro recombination , single stranded binding protein , dna binding site , biochemistry , microbiology and biotechnology , genetics , dna binding protein , molecular cloning , gene , promoter , gene expression , transcription factor
The single‐stranded DNA binding protein (SSB) plays an important role in DNA replication, repair and recombination. To study the biochemical properties of SSB from Mycobacterium tuberculosis ( Mtu SSB), we have used the recently published genome sequence to clone the ssb open reading frame by PCR and have developed an overexpression system. Sequence comparison reveals that the Mtu SSB lacks many of the highly conserved amino acids crucial for the Escherichia coli SSB ( Eco SSB) structure–function relationship. A highly conserved His55, important for homotetramerization of Eco SSB is represented by a leucine in Mtu SSB. Similarly, Trp40, Trp54 and Trp88 of Eco SSB required for stabilizing SSB–DNA complexes are represented by Ile40, Phe54 and Phe88 in Mtu SSB. In addition, a group of positively charged amino acids oriented towards the DNA binding cleft in Eco SSB contains several nonconserved changes in Mtu SSB. We show that in spite of these changes in the primary sequence Mtu SSB is similar to Eco SSB in its biochemical properties. It exists as a tetramer, it has the same minimal size requirement for its efficient binding to DNA and its binding affinity towards DNA oligonucleotides is indistinguishable from that of Eco SSB. Furthermore, Mtu SSB interacts with DNA in at least two distinct modes corresponding to the SSB 35 and SSB 56/65 modes of Eco SSB interaction with DNA. However, Mtu SSB does not form heterotetramers with Eco SSB. Mtu SSB therefore presents us with an interesting system with which to investigate further the role of the conserved amino acids in the biological properties of SSBs.