A functional characterisation of the DNA helicase Ch1R1 in DNA replication and repair

ChlR1 is a DNA helicase implicated in diverse cellular processes including sister chromatid cohesion and DNA replication and repair. However, the mechanism by which ChlR1 participates in these processes is unknown. Data presented in this thesis show that siRNA-mediated depletion of ChlR1 causes increased sensitivity to chemically-induced replication stress. Treatment of ChlR1-depleted cells with hydroxyurea results in increased mono-ubiquitination of PCNA and increased chromatin-associated RPA, indicating stalled DNA replication. Furthermore, ChlR1 is recruited to chromatin following hydroxyurea treatment, supporting a role in the stabilisation of forks during replication stress. Fibroblasts derived from a Warsaw Breakage Syndrome (WABS) patient caused by mutation of ChlR1 (G57R) have both defective sister chromatid cohesion and G2 checkpoint following radiation-induced damage. Complementation with wild-type ChlR1 rescued this mutant phenotype while a known helicase dead mutant of ChlR1 (K50R) or the WABS-associated mutants G57R or ΔK897 did not. However, increased and prolonged Chk1 activation was observed in both K50R and ΔK897 complemented cells after treatment with hydroxyurea while the G57R was comparable to wild-type. These data suggest that the novel WABS mutation (G57R) may retain some wild-type ChlR1 activity and offer important insight into the molecular basis of the WABS phenotype

The cellular DNA helicase ChlR1 regulates chromatin and nuclear matrix attachment of the human papillomavirus type 16 E2 protein and high copy viral genome establishment

In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes

Are they high on steroids? Tailored interventions help improve screening for steroid-induced hyperglycaemia in hospitalised patients.

Steroid-induced hyperglycaemia (SIH) is a common adverse effect in patients both with and without diabetes. This project aimed to improve the screening and diagnosis of SIH by improving the knowledge of healthcare professionals who contribute to the management of SIH in hospitalised patients. Monitoring and diagnosis of SIH were measured in areas of high steroid use in our hospital from May 2016 to January 2017. Several interventions were implemented to improve knowledge and screening for SIH including a staff education programme for nurses, healthcare assistants and doctors. The Trust guidelines for SIH management were updated based on feedback from staff. The changes to the guideline included shortening the document from 14 to 4 pages, incorporating a flowchart summarising the management of SIH and publishing the guideline on the Trust intranet. A questionnaire based on the recommendations of the Joint British Diabetes Societies for SIH was used to assess the change in knowledge pre-intervention and post-intervention. Results showed an increase in junior doctors' knowledge of this topic. Although there was an initial improvement in screening for SIH, this returned to near baseline by the end of the study. This study highlights that screening for SIH can be improved by increasing the knowledge of healthcare staff. However, there is a need for ongoing interventions to sustain this change