Distinct mechanisms of DNA repair in mycobacteria and their implications in attenuation of the pathogen growth

About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. Emergence of drug resistant strains and the protracted treatment strategies have compelled the scientific community to identify newer drug targets, and to develop newer vaccines. In the host macrophages, the bacterium survives within an environment rich in reactive nitrogen and oxygen species capable of damaging its genome. Therefore, for its successful persistence in the host, the pathogen must need robust DNA repair mechanisms. Analysis of M. tuberculosis genome sequence revealed that it lacks mismatch repair pathway suggesting a greater role for other DNA repair pathways such as the nucleotide excision repair, and base excision repair pathways. In this article, we summarize the outcome of research involving these two repair pathways in mycobacteria focusing primarily on our own efforts. Our findings, using Mycobacterium smegmatis model, suggest that deficiency of various DNA repair functions in single or in combinations severely compromises their DNA repair capacity and attenuates their growth under conditions typically encountered in macrophages. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Hypersensitivity of hypoxia grown Mycobacterium smegmatis to DNA damaging agents: Implications of the DNA repair deficiencies in attenuation of mycobacteria

Mycobacteria are an important group of pathogenic bacteria. We generated a series of DNA repair deficient strains of Mycobacterium smegmatis, a model organism, to understand the importance of various DNA repair proteins (UvrB, Ung, UdgB, MutY and Fpg) in survival of the pathogenic strains. Here, we compared tolerance of the M. smegmatis strains to genotoxic stress (ROS and RNI) under aerobic, hypoxic and recovery conditions of growth by monitoring their survival. We show an increased susceptibility of mycobacteria to genotoxic stress under hypoxia. UvrB deficiency led to high susceptibility of M. smegmatis to the DNA damaging agents. Ung was second in importance in strains with single deficiencies. Interestingly, we observed that while deficiency of UdgB had only a minor impact on the strain's susceptibility, its combination with Ung deficiency resulted in severe consequences on the strain's survival under genotoxic stress suggesting a strong interdependence of different DNA repair pathways in safeguarding genomic integrity. Our observations reinforce the possibility of targeting DNA repair processes in mycobacteria for therapeutic intervention during active growth and latency phase of the pathogen. High susceptibility of the UvrB, or the Ung/UdgB deficient strains to genotoxic stress may be exploited in generation of attenuated strains of mycobacteria. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

Processing of DNA double-stranded breaks and intermediates of recombination and repair by saccharomyces cerevisiae Mre11 and its stimulation by Rad50, Xrs2, and Sae2 proteins

Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single-over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.

Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats

Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing. (C) 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a process 5'-3' single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg2+ or Mn2+ and inhibited by Cd2+ suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays, Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interactio between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis

The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB(-) strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung(-)) was found to be similar to 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)ludgB(-)) strain was remarkably high, at similar to 19.6-fold. While CG to TA mutations predominated in the ung(-) and ung(-)/udgb(-) strains, AT to GC mutations were enhanced in the udgB(-) strain. The ung(-)/udgB(-) strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung(-) or udgB(-)). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.

Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

Uracil excision repair in Mycobacterium tuberculosis cell-free extracts

Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. (C) 2011 Elsevier Ltd. All rights reserved.

Structure, function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.

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

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.