Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes

GPs' assessment of patients' readiness to change diet, activity and smoking

BACKGROUND: The Stages of Change Model is increasingly used for lifestyle counselling. In general practice, the use of algorithms to measure stage of change is limited, but for successful counselling it is important to know patients' readiness to change. AIM: To assess the accuracy of the assessment of patients' readiness to change fat consumption, physical activity, and smoking by GPs and general practice registrars. DESIGN OF STUDY: Cross-sectional questionnaire-based survey. SETTING: One hundred and ninety-nine patients at elevated cardiovascular risk aged 40–70 years, 24 GPs, and 21 registrars in Dutch general practices. METHOD: Patients were asked to complete an algorithm to measure their motivation to change fat consumption, physical activity, and smoking. GPs and registrars were given descriptions of the stages of change for the three lifestyles, and were asked to indicate the description that matched their patient. Cohen's κ was calculated as measure of agreement between patients and GPs/registrars. RESULTS: Registrars' patients were younger, and less often overweight and hypertensive than GPs' patients. Cohen's κ for smoking was moderate (0.50, 95% confidence interval [CI] = 0.34 to 0.67 for GPs and 0.47, CI = 0.27 to 0.68 for registrars). Agreement for fat and activity was poor to fair. No differences in accuracy were observed between GPs and registrars (P = 0.07–0.83). CONCLUSIONS: Low accuracy indicates that counselling in general practice is often targeted at the wrong people at the wrong time. Improvements can possibly be achieved by making registration of lifestyle parameters in patient records common practice, and by simply asking patients where they stand in respect to lifestyle change