Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3′–5′ DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA

Evaluation of Surgical Stress Associated with Video-assisted Thoracic Surgery for Esophageal Cancer According to Interleukin-6 Variation in Pleural Cavity Lavage Fluid

Esophagectomy for esophageal cancer is one of the most invasive gastrointestinal surgeries. In 1996, we introduced video-assisted thoracic surgery for esophageal cancer (VATS-E) to reduce surgical stress. In 2010, we started employing artificial pneumothorax (AP) using carbon dioxide gas in VATS-E to further reduce surgical stress. In this study, we evaluated interleukin-6 (IL-6) levels in pleural cavity lavage fluid (PLF) of patients undergoing VATS-E with or without AP, and examined the effect of AP on VATS-E-induced stress. This non-randomized study included patients who underwent VATS-E with or without AP at Showa University Hospital between 2009 and 2013 and from whom PLF could be collected. IL-6 concentrations in PLF were examined before and after the thoracic part of the operation. We compared IL-6 variation, defined as the difference between IL-6 concentrations in PLF before and after the thoracic part of the operation, between patients for whom AP was used and those for whom it was not used. A total of 52 patients were included in the study; 26 underwent VATS-E with AP (group AP), and 26 underwent VATS-E without AP (group NP). IL-6 concentrations in PLF were significantly elevated immediately after the thoracic part of the operation in both groups. IL-6 variation in PLF correlated with both thoracic operative time and blood loss, which were considered practical parameters of surgical stress, and was significantly lower in group AP than in group NP. In conclusion, IL-6 variation in PLF is a useful and sensitive maker of surgical stress during VATS-E. VATS-E with AP is less invasive than VATS-E without AP because AP lowers the perioperative systemic inflammatory response to thoracic surgery

Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη−/−/POLζ−/− cells from the chicken DT40 cell line. POLζ−/− cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη−/−/POLζ−/− cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ−/− cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells

[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Regulation of Gross Chromosomal Rearrangements by Ubiquitin and SUMO Ligases in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery