title: Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange.
abstract: The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, India.
authors.forename: N
authors.initials: N
authors.lastname: Sharadamma
authors.forename: Krishnendu
authors.initials: K
authors.lastname: Khan
authors.forename: Sandeep
authors.initials: S
authors.lastname: Kumar
authors.forename: K Neelakanteshwar
authors.initials: KN
authors.lastname: Patil
authors.forename: Seyed E
authors.initials: SE
authors.lastname: Hasnain
authors.forename: K
authors.initials: K
authors.lastname: Muniyappa
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006657:Histones; C541388:HupB protein, E coli; C510666:HupB protein, Mycobacterium tuberculosis; D050505:Mutant Proteins; D010446:Peptide Fragments; D011089:Polydeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; C490937:hupA protein, E coli; D011938:Rec A Recombinases