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

• Published in: The FEBS journal Vol. 278; no. 18; pp. 3447 - 3462
• Main Authors: Sharadamma, N., Khan, Krishnendu, Kumar, Sandeep, Neelakanteshwar Patil, K., Hasnain, Seyed E., Muniyappa, K.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-09-2011
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cross-Linking Reagents - chemistry; Dimerization; DNA strand exchange; DNA supercoiling; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA, Superhelical - chemistry; DNA, Superhelical - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Histones - chemistry; Histones - genetics; Histones - isolation & purification; Histones - metabolism; HU protein; HupB protein; Kinetics; Mutant Proteins - chemistry; Mutant Proteins - isolation & purification; Mutant Proteins - metabolism; Mycobacterium tuberculosis - metabolism; nucleoid; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Polydeoxyribonucleotides - chemistry; Polydeoxyribonucleotides - metabolism; Protein Interaction Domains and Motifs; Protein Stability; Rec A Recombinases - antagonists & inhibitors; Rec A Recombinases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombination, Genetic; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08267.x

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 Kd values in the nanomolar range. By contrast, the N‐terminal fragment of M. tuberculosis HupB (HupBMtbN) showed diminished DNA‐binding activity, with Kd values in the micromolar range, and the C‐terminal domain was completely devoid of DNA‐binding activity. Unlike HupBMtbN, 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 HupBMtbN, 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.
Structured digital
•
 hupB binds to hupB by cross‐linking study (View interaction)
We show that synergy between the N‐ and C‐terminal domains of Mycobacterium tuberculosis HupB, a two domain architectural protein of the nucleoid, 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.