Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

Abstract

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

“…In one investigation, the transcription of rv3852 was found to be reduced when M. tuberculosis entered a nonreplicating state (32), while in the other, rv3852 expression levels were sevenfold higher in exponential phase than in stationary phase (33). These findings are consistent with those of Sharadamma et al (34), who showed that in vitro Rv3852 binds to DNA replication/repair intermediates and to Holliday junctions, which are mainly generated during replication and exponential growth. Another important feature of the M. tuberculosis Rv3852 protein is its subcellular localization, since it was detected only in the cell membrane and not in the other subcellular compartments or in the culture supernatant (Fig.…”

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

“…Subsequently, annotation of the whole-genome sequence of M. tuberculosis H37Rv revealed the presence of a putative ihf gene in the pathogen (25). Several lines of evidence suggest that E. coli NAPs share relatively low amino acid identity with their counterparts in a wide variety of microorganisms, including M. tuberculosis (2,3,26,27). For example, M. tuberculosis H37Rv ihf (Mtihf) is predicted to encode a single 20-kDa polypeptide compared with two different protein species in E. coli (25).…”

Roles of Nucleoid-Associated Proteins in Stress-Induced Mutagenic Break Repair in Starving Escherichia coli

“…During the transition to stationary phase, Fis drops to undetectable levels and is thought to be absent from starving cells (Ali Azam et al 1999). H-NS functions largely as a transcription repressor, silencing especially genes recently acquired through horizontal transfer (Ueguchi and Mizuno 1993;Perez et al 2008;Sharadamma et al 2010;Dorman 2014). H-NS reaches 40,000 molecules per cell during growth phase but falls sharply on entry into stationary phase (Ali Azam et al 1999).…”