Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.fals

Atrial fibrillation is an independent predictor for in-hospital mortality in patients admitted with SARS-CoV-2 infection.

Background Atrial fibrillation (AF) is the most encountered arrhythmia and has been associated with worse in-hospital outcomes. Objective This study was to determine the incidence of AF in patients hospitalized with coronavirus disease 2019 (COVID-19) as well as its impact on in-hospital mortality. Methods Patients hospitalized with a positive COVID-19 polymerase chain reaction test between March 1 and April 27, 2020, were identified from the common medical record system of 13 Northwell Health hospitals. Natural language processing search algorithms were used to identify and classify AF. Patients were classified as having AF or not. AF was further classified as new-onset AF vs history of AF. Results AF occurred in 1687 of 9564 patients (17.6%). Of those, 1109 patients (65.7%) had new-onset AF. Propensity score matching of 1238 pairs of patients with AF and without AF showed higher in-hospital mortality in the AF group (54.3% vs 37.2%; P \u3c .0001). Within the AF group, propensity score matching of 500 pairs showed higher in-hospital mortality in patients with new-onset AF as compared with those with a history of AF (55.2% vs 46.8%; P = .009). The risk ratio of in-hospital mortality for new-onset AF in patients with sinus rhythm was 1.56 (95% confidence interval 1.42-1.71; P \u3c .0001). The presence of cardiac disease was not associated with a higher risk of in-hospital mortality in patients with AF (P = .1). Conclusion In patients hospitalized with COVID-19, 17.6% experienced AF. AF, particularly new-onset, was an independent predictor of in-hospital mortality

Effect of a Behavioral Nudge on Adoption of an Electronic Health Record-Agnostic Pulmonary Embolism Risk Prediction Tool: A Pilot Cluster Nonrandomized Controlled Trial

OBJECTIVE: Our objective was to determine the feasibility and preliminary efficacy of a behavioral nudge on adoption of a clinical decision support (CDS) tool. MATERIALS AND METHODS: We conducted a pilot cluster nonrandomized controlled trial in 2 Emergency Departments (EDs) at a large academic healthcare system in the New York metropolitan area. We tested 2 versions of a CDS tool for pulmonary embolism (PE) risk assessment developed on a web-based electronic health record-agnostic platform. One version included behavioral nudges incorporated into the user interface. RESULTS: A total of 1527 patient encounters were included in the trial. The CDS tool adoption rate was 31.67%. Adoption was significantly higher for the tool that included behavioral nudges (39.11% vs 20.66%; DISCUSSION: We demonstrated feasibility and preliminary efficacy of a PE risk prediction CDS tool developed using insights from behavioral science. The tool is well-positioned to be tested in a large randomized clinical trial

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA

deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast., we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance

Histone H2A and H2B Are Monoubiquitinated at AID-Targeted Loci

Background: Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined. Methodology/Principal Findings: Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt\u27s B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V(H) and S gamma 3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci. Conclusions/Significance: Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation

Evolution of the Primate APOBEC3A Cytidine Deaminase Gene and Identification of Related Coding Regions

The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1–4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme