Identifying protein–protein interactions in somatic hypermutation

Somatic hypermutation (SHM) in immunoglobulin genes is required for high affinity antibody–antigen binding. Cultured cell systems, mouse model systems, and human genetic deficiencies have been the key players in identifying likely SHM pathways, whereas “pure” biochemical approaches have been far less prominent, but change appears imminent. Here we comment on how, when, and why biochemistry is likely to emerge from the shadows and into the spotlight to elucidate how the somatic mutation of antibody variable (V) regions is generated

DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

In Escherichia coli, damage to the chromosomal DNA induces the SOS response, setting in motion a series of different DNA repair and damage tolerance pathways. DNA polymerase IV (pol IV) is one of three specialised DNA polymerases called into action during the SOS response to help cells tolerate certain types of DNA damage. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. However, the results of several studies indicate that pol IV also acts on other substrates, including single-stranded DNA gaps left behind replisomes that re-initiate replication downstream of a lesion, stalled transcription complexes and recombination intermediates. In this study, we use single-molecule time-lapse microscopy to directly visualize fluorescently labelled pol IV in live cells. We treat cells with the DNA-damaging antibiotic ciprofloxacin, Methylmethane sulfonate (MMS) or ultraviolet light and measure changes in pol IV concentrations and cellular locations through time. We observe that only 5±10% of foci induced by DNA damage form close to replisomes, suggesting that pol IV predominantly carries out non-replisomal functions. The minority of foci that do form close to replisomes exhibit a broad distribution of colocalisation distances, consistent with a significant proportion of pol IV molecules carrying out postreplicative TLS in gaps behind the replisome. Interestingly, the proportion of pol IV foci that form close to replisomes drops dramatically in the period 90±180 min after treatment, despite pol IV concentrations remaining relatively constant. In an SOS-constitutive mutant that expresses high levels of pol IV, few foci are observed in the absence of damage, indicating that within cells access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites

Regulation of mutagenic DNA polymerase V activation in space and time

Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template

Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics

Changing practice in dementia care in the community: developing and testing evidence-based interventions, from timely diagnosis to end of life (EVIDEM)

Background Dementia has an enormous impact on the lives of individuals and families, and on health and social services, and this will increase as the population ages. The needs of people with dementia and their carers for information and support are inadequately addressed at all key points in the illness trajectory. Methods The Unit is working specifically on an evaluation of the impact of the Mental Capacity Act 2005, and will develop practice guidance to enhance concordance with the Act. Phase One of the study has involved baseline interviews with practitioners across a wide range of services to establish knowledge and expectations of the Act, and to consider change processes when new policy and legislation are implemented. Findings Phase 1, involving baseline interviews with 115 practitioners, identified variable knowledge and understanding about the principles of the Act. Phase 2 is exploring everyday decision-making by people with memory problems and their carers

Adefovir Dipivoxil for the Treatment of Hepatitis B e Antigen–Positive Chronic Hepatitis B

BACKGROUND In preclinical and phase 2 studies, adefovir dipivoxil demonstrated potent activity against hepatitis B virus (HBV), including lamivudine-resistant strains. METHODS We randomly assigned 515 patients with chronic hepatitis B who were positive for hepatitis B e antigen (HBeAg) to receive 10 mg of adefovir dipivoxil (172 patients), 30 mg of adefovir dipivoxil (173), or placebo (170) daily for 48 weeks. The primary end point was histologic improvement in the 10-mg group as compared with the placebo group. RESULTS After 48 weeks of treatment, significantly more patients who received 10 mg or 30 mg of adefovir dipivoxil per day than who received placebo had histologic improvement (53 percent [P CONCLUSIONS In patients with HBeAg-positive chronic hepatitis B, 48 weeks of 10 mg or 30 mg of adefovir dipivoxil per day resulted in histologic liver improvement, reduced serum HBV DNA and alanine aminotransferase levels, and increased the rates of HBeAg seroconversion. The 10-mg dose has a favorable risk–benefit profile for long-term treatment. No adefovir-associated resistance mutations were identified in the HBV DNA polymerase gene

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin