SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect types of SUMO conjugate

Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many anti-SUMO MAbs are poorly validated, and antibody matching to detection format is without an evidence base. Here we test the specificity and sensitivity of twenty-four anti-SUMO MAbs towards monomeric and polymeric SUMO1-4 in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, for detecting increased SUMOylation in response to thirteen different stress agents, and as enrichment reagents for SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted wit SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. These data characterize the specificity of twenty-four anti-SUMO antibodies across commonly used assays, creating an enabling resource for the SUMO research community

SUMO, a small, but powerful, regulator of double-strand break repair

The response to a DNA double-stranded break in mammalian cells is a process of sensing and signalling the lesion. It results in halting the cell cycle and local transcription and in the mediation of the DNA repair process itself. The response is launched through a series of post-translational modification signalling events coordinated by phosphorylation and ubiquitination. More recently modifications of proteins by S mall U biquitin-like MO difier (SUMO) isoforms have also been found to be key to coordination of the response (Morris et al. 2009 Nature 462 , 886–890 ( doi:10.1038/nature08593 ); Galanty et al. 2009 Nature 462 , 935–939 ( doi:10.1038/nature08657 )). However our understanding of the role of SUMOylation is slight compared with our growing knowledge of how ubiquitin drives signal amplification and key chromatin interactions. In this review we consider our current knowledge of how SUMO isoforms, SUMO conjugation machinery, SUMO proteases and SUMO-interacting proteins contribute to directing altered chromatin states and to repair-protein kinetics at a double-stranded DNA lesion in mammalian cells. We also consider the gaps in our understanding. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.</jats:p

GSK3β-SCFFBXW7α mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover

IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3β (Glycogen Synthase Kinase 3β) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilised protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear " spent " molecules of IRF1 from transcriptionally engaged target promoters

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Human BRCA1-BARD1 ubiquitin ligase activity counters chromatin barriers to DNA resection

The opposing activities of 53BP1 and BRCA1 influence pathway choice of DNA double-strand break repair. How BRCA1 counters the inhibitory effect of 53BP1 on DNA resection and homologous recombination is unknown. Here we identify the site of BRCA1-BARD1 required for priming ubiquitin transfer from E2~ubiquitin. We demonstrate that BRCA1-BARD1’s ubiquitin ligase activity is required for repositioning 53BP1 on damaged chromatin. We confirm H2A ubiquitylation by BRCA1-BARD1 and show that an H2A-ubiquitin fusion protein promotes DNA resection and repair in BARD1 deficient cells. We show BRCA1-BARD1 function in homologous recombination requires the chromatin remodeler SMARCAD1. SMARCAD1 binding to H2A-ubiquitin, optimal localization to sites of damage and activity in DNA repair requires its ubiquitin-binding CUE domains. SMARCAD1 is required for 53BP1 repositioning and the need for SMARCAD1 in Olaparib or camptothecin resistance is alleviated by 53BP1 loss. Thus BRCA1- BARD1 ligase activity and subsequent SMARCAD1-dependent chromatin remodeling are critical regulators of DNA repair