Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn<SUP>2+</SUP> and DNA-dependent ATPase activity

Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn2+ were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg2+ alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn2+ was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg2+ failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that 32P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn2+-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence

The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites implications for the dispersal of inteins in natural populations

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn2+ and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg2+ alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg2+, PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp122, Asp222, and Glu220) together with immunoprecipitation assays provided compelling evidence to link both the Mg2+- and Mn2+ and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations

Characterization of single-stranded DNA-binding proteins from mycobacteria the carboxyl-terminal 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, whereasEscherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteinsin 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

An investigation on image denoising technique using pixel-component-analysis

This paper authenticates a proficient image denoising scheme with the analysis local pixel coherence. In the dominion of a study about noise and pixel elements in image processing, the influence of the Gaussian effect on image contrast plays a key role. It is found in particular that pixel variations may be vast in some cases which potentially tend to develop irregularities in the image

ANA immunofluorescence versus profile-how well they perform in autoimmune diseases: an analysis of their clinical utility in a tertiary care centre

Background: While Immunofluorescence assay remains the gold standard for the detection of ANA, Immunoprofile by ELISA is being increasingly utilized in view of easy availability and quick results. The study was done to find out whether ANA profile results are comparable with IFA.Methods: About 100 patients who had undergone both immunofluorescence and Immunoprofile were included. Immunofluorescence correlation with profile and their correlation with the disease were analyzed; sensitivity, specificity and predictive values were calculated.Results: ANA was positive in 78% by immunofluorescence; 73% by ANA profile. 22 patients in whom ANA IFA was negative were picked up by ANA profile. 27 patients who were not detected by ANA profile were tested positive by IFA. ANA testing by immuno profile had a sensitivity of 65% with a positive predictive value of 69% when compared with IFA. Immunofluorescence pattern and ANA profile correlated with the diagnosed disease in 63% and 49% respectively. Immunofluorescence pattern correlated with the ANA profile in only 35% of the study subjects. On correlation with the disease, ANA profile scored less compared to ANA-IFA with a sensitivity and specificity of 46% each; positive predictive value of 59%; negative predictive value of 33%. On analysis of individual disease, ANA profile is as good as IFA in SLE and scleroderma in terms of sensitivity. In Sjogren’s syndrome and MCTD, specificity and positive predictive value of ANA profile is high.Conclusions: ANA IFA performs better than immunoprofile in the diagnosis of autoimmune diseases

Homologous recombination in mycobacteria

In recent years, considerable effort and resources have been expended to develop targeted gene delivery methods, and generation of auxotrophic mutants of mycobacteria. The results of these studies suggest that mycobacteria exhibit a wide range of recombination rates, which vary from loci to loci. Here we review the methods developed for allele exchange and targeted gene disruption as well as the mechanistic aspects of homologous recombination in mycobacteria. The results of whole genome, functional and structural analyses of Mycobacterium tuberculosis and Mycobacterium smegmatis RecA and SSB proteins provide insights into variations of the prototypic Escherichia coli paradigm. This variation of a common theme might allow mycobacteria to function in their natural but complex physiological environments

Resourcing resilience: Social protection for HIV prevention amongst children and adolescents in Eastern and Southern Africa

Adolescents are the only age group with growing AIDS-related morbidity and mortality in Eastern and Southern Africa, making HIV prevention research among this population an urgent priority. Structural deprivations are key drivers of adolescent HIV infection in this region. Biomedical interventions must be combined with behavioural and social interventions to alleviate the socio-structural determinants of HIV infection. There is growing evidence that social protection has the potential to reduce the risk of HIV infection among children and adolescents. This research combined expert consultations with a rigorous review of academic and policy literature on the effectiveness of social protection for HIV prevention among children and adolescents, including prevention for those already HIV-positive. The study had three goals: (i) assess the evidence on the effectiveness of social protection for HIV prevention, (ii) consider key challenges to implementing social protection programmes that promote HIV prevention, and (iii) identify critical research gaps in social protection and HIV prevention, in Eastern and Southern Africa. Causal pathways of inequality, poverty, gender and HIV risk require flexible and responsive social protection mechanisms. Results confirmed that HIV-inclusive child- and adolescent-sensitive social protection has the potential to interrupt risk pathways to HIV infection and foster resilience. In particular, empirical evidence (literature and expert feedback) detailed the effectiveness of combination social protection particularly cash/in-kind components combined with “care” and “capability” among children and adolescents. Social protection programmes should be dynamic and flexible, and consider age, gender, HIV-related stigma, and context, including cultural norms, which offer opportunities to improve programmatic coverage, reach and uptake. Effective HIV prevention also requires integrated social protection policies, developed through strong national government ownership and leadership. Future research should explore which combinations of social protection work for sub-groups of children and adolescents, particularly those living with HIV

Large-scale sequencing identifies multiple genes and rare variants associated with Crohn’s disease susceptibility

peer reviewe

Structural and functional characteristics of homing endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases