hMMS2 serves a redundant role in human PCNA polyubiquitination

<p>Abstract</p> <p>Background</p> <p>In yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.</p> <p>Results</p> <p>In this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.</p> <p>Conclusion</p> <p>Our results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.</p

The SUMO Isopeptidase Ulp2p Is Required to Prevent Recombination-Induced Chromosome Segregation Lethality following DNA Replication Stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress

Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness

UEV-1 Is an Ubiquitin-Conjugating Enzyme Variant That Regulates Glutamate Receptor Trafficking in C. elegans Neurons

The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect—a decrease in spontaneous reversals in locomotion—consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking

Poststroke anxiety is prevalent at the population level, especially among socially deprived and younger age community stroke survivors

BACKGROUND: Most studies of poststroke anxiety prevalence are hospital based, so knowledge of anxiety in community stroke survivors is limited. Few studies address the association between poststroke anxiety and patient age. No study has explored the relationship between poststroke anxiety prevalence and social deprivation. AIMS: This study aims to describe population level prevalence data of poststroke anxiety and to explore association of poststroke anxiety prevalence with patient age, gender, and social deprivation. METHODS: Observational study of 3831 community stroke survivors attending general practice reviews from April 1, 2009 to March 31, 2010 in Greater Glasgow, United Kingdom. Univariate and multivariate analyses investigated the association between poststroke anxiety prevalence (Hospital Anxiety and Depression Scale: anxiety sub-scale [HADS-A]), age, gender, and deprivation variables. RESULTS: Six hundred eighteen (16·1%) of 3831 community-dwelling stroke survivors had definite abnormal mood on HADS-A (≥11), with 952 (31·5%) scoring ≥8. Sixty-five (35·5%) of stroke survivors aged under 50 years had definite abnormal mood on HADS-A compared with 59 (7·2%) of over 80 year olds. Three hundred forty (19·8%) of women had definite abnormal mood on HADS-A compared with 278 (13·1%) of men. Three hundred seventy-two (22·6%) of most deprived stroke survivors had definite abnormal mood on HADS-A compared with 49 (7·6%) of least deprived. Age, gender, and social deprivation all contributed significantly to HADS-A score variance. CONCLUSION: Using a conservative HADS-A cutoff, a high prevalence of definite abnormal anxiety in community stroke survivors is observed. This prevalence increases markedly in younger and more socially deprived stroke survivors