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.

HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

“…Previously, the N-terminal domain (1 to 95 aa) has been shown to interact with DNA, albeit at a lower efficiency compared to the full-length HupB, leading to speculation that the C-terminal domain acts as a DNA clasp (43). We, however, observed that the N-terminal domain (1 to 108 aa) used in the present study demonstrated a higher affinity to dsDNA compared to a shorter fragment of the N-terminal domain (1 to 95 aa) used previously (42). This could partially be attributed to the length of the N-terminal domain and shows that the residues beyond the HU fold may be critical for N-HupB-DNA interaction.…”

“…6A. GST-N-HupB showed efficient interaction with double-stranded DNA (dsDNA) and with a higher affinity than previously reported (42). The promoter region of the Rv1230c gene, another member of CRP regulon, also displayed a similar binding affinity with HupB (data not shown).…”

“…The EMSA was carried out with increasing concentrations of N-HupB (10 to 100 nM) and the promoter region of the Rv0884c gene. Notably, M. tuberculosis HupB and a shorter version of its N-terminal domain (1 to 95 amino acids [aa]) have been previously reported to interact with various forms of DNA in a non-sequence-specific manner (42,43). Hence, the DNA probe used in the present study was selected randomly and was the promoter region previously shown to be targeted by cyclic AMP receptor protein (CRP) (44).…”

Defining the Functionally Important Domain and Amino Acid Residues in Mycobacterium tuberculosis Integration Host Factor for Genome Stability, DNA Binding, and Integrative Recombination

“…Homologous NAPs are widely distributed across bacterial species, including Mycobacteria, Streptomyces, Rhodococcus, and Corynebacteria (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24); however, they are far less well characterized. The focus of the present study is mainly on the characterization of the IHF of M. tuberculosis; consequently, the existing literature relevant to IHF is discussed here.…”

The abundant and essential HU proteins in Deinococcus deserti and Deinococcus radiodurans are translated from leaderless mRNA

“…The N-and C-terminal extensions in HU resemble the (S/T)PKK repeats in the C-terminal domain of eukaryotic histone H1, which are involved in DNA compaction (Bharath et al, 2002). In vitro experiments have shown that the Mycobacterium HU extensions have an important contribution to DNA binding, modulate the DNA binding specificities compared with the corresponding truncated proteins consisting of the conserved core HU domain, and may have a role in DNA protection and compaction (Grove, 2011;Kumar et al, 2010;Mukherjee et al, 2008;Sharadamma et al, 2011). In vitro DNA-binding experiments have also been performed with recombinant D. radiodurans HU proteins (i.e.…”