The ubiquitin E3/E4 ligase, UBE4A, fine-tunes protein ubiquitylation and accumulation at sites of DNA damage facilitating double-strand break repair

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning - the E3/E4 ubiquitin ligase, UBE4A. UBE4A’s recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end-resection at DSBs, and its abrogation leads to up-regulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning of the complex DDR network for accurate and balanced execution of DSB repai

Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity

IntroductionThe success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance.MethodsWe report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls.ResultsIn contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients.DiscussionThese features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair. © 2014 Nakajima et al

A first update on mapping the human genetic architecture of COVID-19

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The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

<div><p>Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used <i>Arabidopsis thaliana</i> mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus <i>Botrytis cinerea</i>, which infects a variety of hosts, with the Brassicaceae-specific fungus <i>Alternaria brassicicola</i>. <i>B. cinerea</i> isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while <i>A. brassicicola</i> was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that <i>B. cinerea</i> stimulates the accumulation of glucosinolates to a greater extent than <i>A. brassicicola</i>. In our work with <i>A. brassicicola</i>, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, <i>in vivo</i>, hydrolysis products of indole glucosinolates were found to be involved in defense against <i>B. cinerea</i>, but not in the host response to <i>A. brassicicola</i>. We suggest that the Brassicaceae-specialist <i>A. brassicicola</i> has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist <i>B. cinerea</i> are more sensitive to these phytochemicals.</p></div

Effects of indole glucosinolate and camalexin on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants <i>cyp79B2/B3</i> and <i>pad3</i>, which have altered total glucosinolate and/or camalexin content, and their corresponding wild-type background (Col-0) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation (upper and middle panels) with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i> (lower panel). Average lesion sizes from 30 leaves of each genotype are presented along with and the standard error of each average. All numbers are presented as the relative percentage to their corresponding background wild-type. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Impact of aliphatic glucosinolate on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from plants containing the double-knockout <i>myb28 myb29</i> (<i>myb28/29</i>) expressed against the Col-0 background (A) and plants in which <i>MYB29^OXP</i> (MYB29) and <i>MYB34^OXP</i> (MYB34) were expressed against the Ler background (B) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion sizes from 10 to 17 leaves of each genotype are presented together with the standard errors for each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters or asterisks above the columns indicate statistically significant differences at <i>P</i>>0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of glucosinolate-turnover products on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from wild-type, <i>pen2</i>, <i>cyp81F2</i> and <i>pen2/cyp81F2</i> plants were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 <i>B. cinerea</i> isolate (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion areas for 30 leaves of each genotype are presented together with the standard error for each average. All numbers are presented as the relative lesion size as compared to the lesions observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of glucosinolate-breakdown products on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants with altered total glucosinolate-breakdown product contents and containing different relative amounts of the different type of products were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 isolate of <i>B. cinerea</i> (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h or 120 to 192 h post-inoculation (<i>B. cinerea</i> and <i>A. brassicicola</i>, respectively) on leaves from <i>tgg1-3/tgg2-1</i> (<i>tgg1/2</i>) plants, <i>35S:ESP</i> plants, the wild-types Col-0 and Ler and the triple mutant <i>35:ESP/tgg1-3/tgg2-1</i> (<i>tgg1/2:ESP</i>). (All mutations were expressed against the Col-0 background.) Average lesion areas from 15 to 30 leaves of each genotype are presented together with the standard error of each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p