Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.

Infection after primary total hip arthroplasty.

The number of primary total hip arthroplasties (THAs) performed in the United States each year continues to climb, as does the incidence of infectious complications. The changing profile of antibiotic-resistant bacteria has made preventing and treating primary THA infections increasingly complex. The goal of this review was to summarize (1) the published data concerning the risk of surgical site infection (SSI) after primary THA by type of bacteria and (2) the effect of potentially modifying factors. The Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, EMBASE, Web of Science, and PubMed were searched. Studies dated between 2001 and 2011 examining primary THA in adults were included. Meta-analysis of the collected data was performed. The pooled SSI rate was 2.5% (95% confidence interval [Cl], 1.4%-4.4%; P<.001; n=28,883). The pooled deep prosthetic joint infection (PJI) rate was 0.9% (95% Cl, 0.4%-2.2%; P<.001; n=28,883). The pooled rate of methicillin-resistant Staphylococcus aureus SSI was 0.5% (95% Cl, 0.2%-1.5%; P<.001; n=26,703). This is approximately 20% of all SSI cases. The pooled rate of intraoperative bacterial wound contamination was 16.9% (95% Cl, 6.6%-36.8%; P=.003; n=2180). All these results had significant heterogeneity. The postoperative risk of SSI was significantly associated with intraoperative bacterial surgical wound contamination (pooled rate ratio, 2.5; 95% Cl, 1.4%-4.6%; P=.001; n=19,049).