Cloning, over-expression and biochemical characterization of the single-stranded DNA binding protein from Mycobacterium tuberculosis.
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 (MtuSSB), 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 MtuSSB lacks many of the highly conserved amino acids crucial for the Escherichia coli SSB (EcoSSB) structure-function relationship. A highly conserved His55, important for homotetramerization of EcoSSB is represented by a leucine in MtuSSB. Similarly, Trp40, Trp54 and Trp88 of EcoSSB required for stabilizing SSB-DNA complexes are represented by Ile40, Phe54 and Phe88 in MtuSSB. In addition, a group of positively charged amino acids oriented towards the DNA binding cleft in EcoSSB contains several nonconserved changes in MtuSSB. We show that in spite of these changes in the primary sequence MtuSSB is similar to EcoSSB 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 EcoSSB. Furthermore, MtuSSB interacts with DNA in at least two distinct modes corresponding to the SSB35 and SSB56/65 modes of EcoSSB interaction with DNA. However, MtuSSB does not form heterotetramers with EcoSSB. MtuSSB 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.
Purnapatre
K
K
Department of Microbiology, Indian Institute of Science, Bangalore, India.
Varshney
U
U
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C121721:SSB protein, Mycobacterium tuberculosis; D014364:Tryptophan