Cryo-EM structure of translesion DNA synthesis polymerase ζ with a base pair mismatch

[EN] The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an “open” conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes.This work was funded by grants R01-GM124047 (A.K.A and L.P) and R35-GM13170 (A.K.A) from the National Institutes of Health (NIH). I.U.-B was supported by a grant PID2019-104423GB-I00/AEI/10.13039/501100011033 from the Spanish State Research Agency and by the Basque Excellence Research Centre program. Most of the cryo-EM work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310), with additional support from Agouron Institute (F00316), NIH (OD019994) and NIH (RR029300). Computing resources needed for this work were provided in part by the High Performance Computing facility of the Icahn School of Medicine at Mount Sinai. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311

Reciprocal Interactions of Pit1 and GATA2 Mediate Signaling Gradient–Induced Determination of Pituitary Cell Types

AbstractThe mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type–specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding–independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding–dependent and –independent actions of abundant determining factors contribute to generate distinct cell phenotypes

Brd4 and JMJD6-Associated Anti-Pause Enhancers in Regulation of Transcriptional Pause Release

论文以我校为共同通讯单位，刘文教授为共同第一和通讯作者。论文对去甲基化酶蛋白家族中的成员JMJD6在基因转录中的新颖分子作用机制进行了阐述。研究表明JMJD6和另一热门表观遗传学调控子Brd4通过一种全新的“远程调控”模式调节RNA聚合酶II(PolII)在基因启动子附近停顿后重新激活(promoter-proximal　pause　release)和基因转录延伸过程（transcriptional　elongation）。文章首次将相关的远程调控基因组增强子序列命名为“抗停顿增强子”(Anti-pause　enhancers)。值得一提的是，JMJD6和Brd4与多种人类癌症和其它疾病紧密相联，例如乳腺癌的发生发展。研究成果极大地提高了对基因转录延伸机制的理解，同时也为治疗癌症等相关疾病提供了新的理论依据和思路指导，在理论研究及应用方面意义重大。 　　刘文教授是药学院2013年重点引进的优秀人才，因其出色的学术能力和科研潜力，经学院申报，入选当年的中组部“青年千人计划”和“闽江学者”。刘文教授一直致力于从事表观遗传学调控子以及核受体的分子作用机制及其在癌症等重大疾病发生发展中的应用研究，论文多次发表在Nature，Cell,CancerCell，MolecularCell,PNAS等国际权威科学期刊。Distal enhancers characterized by the H3K4me mark play critical roles in developm ental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA polymerase II (Pol II) promoter-proximal pause release remain poor lyinves tigated . Here, we report that a unique cohort of jumo njiC-domain-con taining protein 6 (JMJD 6) and bromodomain-containing protein 4 (Brd4) cobound distal enhancers , termed a ntip ause e nhance rs (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range in teractions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me, which is directly read by 7SK snR NA,and decappi ng/demet hylation of 7SK snR NA,ensurin g the dis missal of the 7SK snRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes.The functions of JM JD6/Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis, and disease

Topology of Type II REases revisited; structural classes and the common conserved core

Type II restriction endonucleases (REases) are deoxyribonucleases that cleave DNA sequences with remarkable specificity. Type II REases are highly divergent in sequence as well as in topology, i.e. the connectivity of secondary structure elements. A widely held assumption is that a structural core of five β-strands flanked by two α-helices is common to these enzymes. We introduce a systematic procedure to enumerate secondary structure elements in an unambiguous and reproducible way, and use it to analyze the currently available X-ray structures of Type II REases. Based on this analysis, we propose an alternative definition of the core, which we term the αβα-core. The αβα-core includes the most frequently observed secondary structure elements and is not a sandwich, as it consists of a five-strand β-sheet and two α-helices on the same face of the β-sheet. We use the αβα-core connectivity as a basis for grouping the Type II REases into distinct structural classes. In these new structural classes, the connectivity correlates with the angles between the secondary structure elements and with the cleavage patterns of the REases. We show that there exists a substructure of the αβα-core, namely a common conserved core, ccc, defined here as one α-helix and four β-strands common to all Type II REase of known structure

Structure of Human DNA Polymerase κ Inserting dATP Opposite an 8-OxoG DNA Lesion

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ\u27s preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries