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Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

ABSTRACT: The bacterial chromosomal DNA is folded into a compact structure called as 'nucleoid' so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like "PAKK" motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.

SUBMITTER: Mishra A

PROVIDER: S-EPMC3724605 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

Mishra Arpit A Vij Manika M Kumar Dhirendra D Taneja Vibha V Mondal Anupam Kumar AK Bothra Ankur A Rao Vivek V Ganguli Munia M Taneja Bhupesh B

PloS one 20130726 7

The bacterial chromosomal DNA is folded into a compact structure called as 'nucleoid' so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NA ...[more]

PMID: 23922883

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Project description:The integration host factor of Mycobacterium tuberculosis (mIHF) consists of a single polypeptide chain, the product of the ihf gene. We previously revealed that mIHF is a novel member of a new class of nucleoid-associated proteins that have important roles in DNA damage response, nucleoid compaction, and integrative recombination. The mIHF contains a region of 86 amino acids at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF. However, the functional significance of an extra 86-amino-acid region in the full-length protein remains unknown. Here, we report the structure/function relationship of the DNA-binding and integrative recombination-stimulating activity of mIHF. Deletion mutagenesis showed that an extra 86-amino-acid region at the N terminus is dispensable; the C-terminal region possesses the sequences essential for its known biological functions, including the ability to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents, DNA binding, DNA multimerization-circularization, and stimulation of phage L5 integrase-catalyzed integrative recombination. Single and double alanine substitutions at positions Arg170 and Arg171, located at the mIHF DNA-binding site, abrogated its capacity to suppress the sensitivity of E. coli ΔihfA and ΔihfB cells to DNA-damaging agents. The variants encoded by these mutant alleles failed to bind DNA and stimulate integrative recombination. Interestingly, the DNA-binding activity of the mIHF-R173A variant remained largely unaffected; however, it was unable to stimulate integrative recombination, thus revealing a separation-of-function allele of mIHF. The functional and structural characterization of this separation-of-function allele of mIHF could reveal previously unknown functions of IHF.IMPORTANCE The integration host factor of Mycobacterium tuberculosis is a novel nucleoid-associated protein. mIHF plays a vital role in DNA damage response, nucleoid compaction, and integrative recombination. Intriguingly, mIHF contains an extra 86-amino-acid region at its N terminus, absent from both α- and β-subunits of Escherichia coli IHF, whose functional significance is unknown. Furthermore, a triad of arginine residues located at the mIHF-DNA interface have been implicated in a range of its functions. Here, we reveal the roles of N- and C-terminal regions of mIHF and the individual residues in the Arg triad for their ability to provide protection in vivo against DNA damage, bind DNA, and stimulate integrase-catalyzed site-specific recombination.

Project description:Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

Dataset Information

Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

Publications

Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

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