Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys 1226 to cytosine (Cyt) C6, methyl transfer from S-adenosyl-L-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys 1226 attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.H uman DNA methyltransferases (DNMTs) catalyze the formation of 5-methylcytosine (5mC) at CpG sites on DNA, a key epigenetic mark present in the human genome (1). DNA methylation is involved in transcriptional silencing, cellular differentiation, genomic imprinting, and X-chromosome inactivation. In addition, hypermethylation of CpG islands at gene promoter regions has been associated with carcinogenesis (2). Maintenance of DNA methylation patterns is conducted by human DNMT1, a multidomain protein of 1,616 amino acids. The C-terminal methyltransferase domain shows sequence similarities to the bacterial methyltransferases (3). Crystal structures of mouse and human DNMT1 complexed with different substrates have provided a structural basis for DNMT1-mediated maintenance DNA methylation (4, 5). Domain interactions and large conformational changes are responsible for properly positioning hemimethylated DNA within the active site and catalyze methyl transfer from S-adenosyl-L-methionine (SAM) to DNA. Site-directed mutations have offered insights into the structure-function relationship of DNMTs (6, 7), but their transition state (TS) structures have remained unknown. DNMT1 has been proposed to follow a catalytic mechanism shared by bacterial DNA-(cytosine C5)-methyltransferases (4, 8-10): nucleophilic attack of cytosine (Cyt) C6 by Cys 1226 of DNMT1, methyl transfer from SAM to Cyt C5, and β-elimination of H5 to produce 5mC in the final step (Fig. 1). Recent quantum mechanics (QM)/molecular mechanics (MM) and molecular dynamics (MD) simulations of the bacterial M.HhaI methyltransferase suggested that Cys 1226 attack is concerted with methyl transfer (11,12), and that β-elimination of H5 is the rate-limiting step (12). The combination of kinetic isotope effects (KIEs) and computational chemistry can test predicted reaction mechanisms and can provide a model of the T...
BackgroundThe rapid spread of Clostridium difficile NAP1/BI/027 (C. difficile 027) has become one of the leading threats of healthcare-associated infections worldwide. However, C. difficile 027 infections have been rarely reported in Asia, particularly in China.ResultsIn this study, we identified a rare C. difficile bloodstream infection (BSI) from three isolates of a patient during repeated hospital admission. This finding triggered a retrospective epidemiological study to scan all cases and strains emerged from this ward during the past three years. Using medical personnel interviews, medical record reviews and the genomic epidemiology, two outbreaks in 2012 and 2013–2014 were identified. Through using whole genome sequencing, we succeeded to trace the origin of the BSI strain. Surprisingly, we found the genome sequences were similar to C. difficile 027 strain R20291, indicating the occurrence of a rare C. difficile 027 strain in China. Integrated epidemiological investigation and whole genome sequencing of all strains, we constructed a nosocomial transmission map of these two C. difficile 027 outbreaks and traced the origin of the infection.ConclusionsBy genome sequencing, spatio-temporal analysis and field epidemiology investigation, we can estimate their complex transform network and reveal the possible modes of transmission in this ward. Based on their genetic diversity, we can assume that the toilets, bathroom, and janitor’s equipment room may be contaminated area, which may be suggested to improve infection control measures in the following health care.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2708-0) contains supplementary material, which is available to authorized users.
The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens.
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