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.

Base excision and nucleotide excision repair pathways in mycobacteria

About a third of the human population is estimated to be infected with Mycobacterium tuberculosis. The bacterium displays an excellent adaptability to survive within the host macrophages. As the reactive environment of macrophages is capable of inducing DNA damage, the ability of the pathogen to safeguard its DNA against the damage is of paramount significance for its survival within the host. Analysis of the genome sequence has provided important insights into the DNA repair machinery of the pathogen, and the studies on DNA repair in mycobacteria have gained momentum in the past few years. The studies have revealed considerable differences in the mycobacterial DNA repair machinery when compared with those of the other bacteria. This review article focuses especially on the aspects of base excision, and nucleotide excision repair pathways in mycobacteria. (C) 2011 Elsevier Ltd. All rights reserved.

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1. cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1. sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.

Rapid transfection assay for screening mutagens and carcinogens

Testing for mutagenicity and carcinogenicity has become an integral part of the toxicological evaluation of drugs and chemicals. Standard carcinogenicity tests in vivo require both large numbers of animals and prolonged experiments. To circumvent these problems, several rapid tests have been developed for preliminary screening of mutagens and carcinogens in vitro. Ames and his associates, the first to develop a mutation test, used mutant strains of Salmonella typhimurium [1]. Mutation tests with Escherichia coli, Bacillus subtilis, Neurospora crassa and Saccharomyces cerevisiae, and DNA-repair tests with E. coli and B. subtilis, have been developed. Cytogenetic assays, in vivo as well as in vitro, in both plant and animal systems, are also used to detect potential mutagens and carcinogens. Transfection is inhibited by base mutation, cleavage of DNA, loss of cohesive ends, interaction with histones, spermidine, nalidixic acid, etc. [3]. The efficiency of transfection is affected by temperature, DNA structure and the condition of the competence of the recipient cells [3]. Transfection assays with phages MS: RNA and ~i, x 174-DNA have been reported [15]. A fast and easy transfection assay using colitis bacteriophage DNA is reported in this communication.

A distinct physiological role of MutY in mutation prevention in mycobacteria

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-B-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG: A, 8-oxoG: G or 8-oxoG: T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India

Background & objectives: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677C -> T and 1298 A -> C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C -> T and 1298 A -> C and risk of colorectal cancer (CRC).Methods: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). Results: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74,25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A -> C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45(95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18(95% CI 0.52-2.64, P =0.697).Interpretation & conclusion: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C -> T and 1298 A -> C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A -> C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. Materials and Methods: Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method. Results: Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A→C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48- 2.45, p = 0.851) and OR for A1298C was 1.29(95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p =0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58 -6.52, p = 0.286). Conclusion: The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C→T and 1298 A→C gene polymorphisms and risk of ALL, which may be due to the small sample size.

Structural alteration from non-B to B-form could reflect DNase I hypersensitivity

Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.