Histone Deposition Protein Asf1 Maintains DNA Replisome Integrity and Interacts with Replication Factor C
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Regulates Cellular Telomerase Activity by Methylation of TERT Promoter
www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 5), pp: 7977-7988 Research Paper Tianshengyuan-1 (TSY-1) regulates cellular Telomerase activity by methylation of TERT promoter Weibo Yu1, Xiaotian Qin2, Yusheng Jin1, Yawei Li2, Chintda Santiskulvong3, Victor Vu1, Gang Zeng4,5, Zuofeng Zhang6, Michelle Chow1, Jianyu Rao1,5 1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 2Beijing Boyuantaihe Biological Technology Co., Ltd., Beijing, China 3Genomics Core, Cedars-Sinai Medical Center, Los Angeles, CA, USA 4Department of Urology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA 5Jonsson Comprehensive Cancer Center, University of California at Los Angeles, Los Angeles, CA, USA 6Department of Epidemiology, School of Public Health, University of California at Los Angeles, Los Angeles, CA, USA Correspondence to: Jianyu Rao, email: [email protected] Keywords: TSY-1, hematopoietic cells, Telomerase, TERT, methylation Received: September 08, 2016 Accepted: November 24, 2016 Published: December 15, 2016 ABSTRACT Telomere and Telomerase have recently been explored as anti-aging and anti- cancer drug targets with only limited success. Previously we showed that the Chinese herbal medicine Tianshengyuan-1 (TSY-1), an agent used to treat bone marrow deficiency, has a profound effect on stimulating Telomerase activity in hematopoietic cells. Here, the mechanism of TSY-1 on cellular Telomerase activity was further investigated using HL60, a promyelocytic leukemia cell line, normal peripheral blood mononuclear cells, and CD34+ hematopoietic stem cells derived from umbilical cord blood. TSY-1 increases Telomerase activity in normal peripheral blood mononuclear cells and CD34+ hematopoietic stem cells with innately low Telomerase activity but decreases Telomerase activity in HL60 cells with high intrinsic Telomerase activity, both in a dose-response manner. -
A New Class of Disordered Elements Controls DNA Replication Through
RESEARCH ARTICLE A new class of disordered elements controls DNA replication through initiator self-assembly Matthew W Parker1,2, Maren Bell2, Mustafa Mir2, Jonchee A Kao2, Xavier Darzacq2, Michael R Botchan2*, James M Berger1* 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, United States; 2Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States Abstract The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co- assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter- origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co- assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization. DOI: https://doi.org/10.7554/eLife.48562.001 *For -
Anti-RFC3 (GW21946F)
3050 Spruce Street, Saint Louis, MO 63103 USA Tel: (800) 521-8956 (314) 771-5765 Fax: (800) 325-5052 (314) 771-5757 email: [email protected] Product Information Anti-RFC3 antibody produced in chicken, affinity isolated antibody Catalog Number GW21946F Formerly listed as GenWay Catalog Number 15-288-21946F, Replication factor C subunit 3 Antibody. – Storage Temperature Store at 20 °C The product is a clear, colorless solution in phosphate buffered saline, pH 7.2, containing 0.02% sodium azide. Synonyms: Replication factor C 3 isoform 1, Replication factor C 38 kDa subunit; RFC38; Activator 1 38 kDa subunit; Species Reactivity: Human, rat A1 38 kDa subunit; RF-C 38 kDa subunit Tested Applications: ELISA, WB Product Description Recommended Dilutions: Recommended starting dilution The elongation of primed DNA templates by DNA for Western blot analysis is 1:500, for tissue or cell staining polymerase delta and epsilon requires the action of the 1:200. accessory proteins proliferating cell nuclear antigen (PCNA) and activator 1. Note: Optimal concentrations and conditions for each application should be determined by the user. NCBI Accession number: NP_002906.1 Swiss Prot Accession number: P40938 Precautions and Disclaimer This product is for R&D use only, not for drug, household, or Gene Information: Human .. RFC3 (5983) other uses. Due to the sodium azide content a material Immunogen: Recombinant protein Replication factor C 3 safety data sheet (MSDS) for this product has been sent to isoform 1 the attention of the safety officer of your institution. Please consult the Material Safety Data Sheet for information Immunogen Sequence: GI # 4506489, sequence 1 - 356 regarding hazards and safe handling practices. -
Structure of the Human Clamp Loader Reveals an Autoinhibited Conformation of a Substrate-Bound AAA+ Switch
Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch Christl Gaubitza,1, Xingchen Liua,b,1, Joseph Magrinoa,b, Nicholas P. Stonea, Jacob Landecka,b, Mark Hedglinc, and Brian A. Kelcha,2 aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester MA 01605; bGraduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester MA 01605; and cDepartment of Chemistry, The Pennsylvania State University, University Park, PA 16802 Edited by Michael E. O’Donnell, HHMI and Rockefeller University, New York, NY, and approved July 27, 2020 (received for review April 20, 2020) DNA replication requires the sliding clamp, a ring-shaped protein areflexia syndrome (15), Hutchinson–Gilford progeria syn- complex that encircles DNA, where it acts as an essential cofactor drome (16), and in the replication of some viruses (17–19). It for DNA polymerases and other proteins. The sliding clamp needs is unknown whether loading by RFC contributes to PARD to be opened and installed onto DNA by a clamp loader ATPase of disease. the AAA+ family. The human clamp loader replication factor C Clamp loaders are members of the AAA+ family of ATPases (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) (ATPases associated with various cellular activities), a large are both essential and play critical roles in several diseases. De- protein family that uses the chemical energy of adenosine 5′- spite decades of study, no structure of human RFC has been re- triphosphate (ATP) to generate mechanical force (20). Most solved. Here, we report the structure of human RFC bound to AAA+ proteins form hexameric motors that use an undulating PCNA by cryogenic electron microscopy to an overall resolution ∼ spiral staircase mechanism to processively translocate a substrate of 3.4 Å. -
Molecular Anatomy and Regulation of a Stable Replisome at a Paused Eukaryotic DNA Replication Fork
Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork Arturo Calzada,1,2,3 Ben Hodgson,1,3 Masato Kanemaki,1 Avelino Bueno,2 and Karim Labib1,4 1Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom; 2Cancer Research Institute, University of Salamanca/CSIC, 37007 Salamanca, Spain Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM–Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM–Cdc45, GINS, and DNA polymerases ␣ and and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently. -
Anti-RFC3 (Internal Region) Polyclonal Antibody (DPABH-26536) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
Anti-RFC3 (internal region) polyclonal antibody (DPABH-26536) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Antigen Description The elongation of primed DNA templates by DNA polymerase delta and epsilon requires the action of the accessory proteins proliferating cell nuclear antigen (PCNA) and activator 1. Immunogen Synthetic peptide derived from an internal sequence of Human RFC3. Isotype IgG Source/Host Rabbit Species Reactivity Human Purification Immunogen affinity purified Conjugate Unconjugated Applications WB, IHC-P Format Liquid Size 100 μg Buffer pH: 7.40; Constituents: 49% PBS, 50% Glycerol, 0.88% Sodium chloride. Note: PBS is without Mg2+ and Ca2+ Preservative 0.02% Sodium Azide Storage Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. GENE INFORMATION Gene Name RFC3 replication factor C (activator 1) 3, 38kDa [ Homo sapiens ] Official Symbol RFC3 Synonyms RFC3; replication factor C (activator 1) 3, 38kDa; replication factor C (activator 1) 3 (38kD); replication factor C subunit 3; A1 38 kDa subunit; MGC5276; RFC; 38 kD subunit; RFC38; RFC, 38 kD subunit; RF-C 38 kDa subunit; activator 1 subunit 3; activator 1 38 kDa subunit; replication factor C 38 kDa subunit; 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved Entrez Gene ID 5983 Protein Refseq NP_002906 UniProt ID A0A024RDQ8 Chromosome Location 13q13.2 Pathway Activation of ATR in response to replication stress; BRCA1-associated genome surveillance complex (BASC); Cell Cycle; Cell Cycle Checkpoints; Cell Cycle, Mitotic; Chromosome Maintenance; DNA Repair Function ATP binding; contributes_to ATPase activity; contributes_to DNA binding; DNA clamp loader activity; nucleotide binding; protein binding; 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 2 © Creative Diagnostics All Rights Reserved. -
Supplementary Table S1. Correlation Between the Mutant P53-Interacting Partners and PTTG3P, PTTG1 and PTTG2, Based on Data from Starbase V3.0 Database
Supplementary Table S1. Correlation between the mutant p53-interacting partners and PTTG3P, PTTG1 and PTTG2, based on data from StarBase v3.0 database. PTTG3P PTTG1 PTTG2 Gene ID Coefficient-R p-value Coefficient-R p-value Coefficient-R p-value NF-YA ENSG00000001167 −0.077 8.59e-2 −0.210 2.09e-6 −0.122 6.23e-3 NF-YB ENSG00000120837 0.176 7.12e-5 0.227 2.82e-7 0.094 3.59e-2 NF-YC ENSG00000066136 0.124 5.45e-3 0.124 5.40e-3 0.051 2.51e-1 Sp1 ENSG00000185591 −0.014 7.50e-1 −0.201 5.82e-6 −0.072 1.07e-1 Ets-1 ENSG00000134954 −0.096 3.14e-2 −0.257 4.83e-9 0.034 4.46e-1 VDR ENSG00000111424 −0.091 4.10e-2 −0.216 1.03e-6 0.014 7.48e-1 SREBP-2 ENSG00000198911 −0.064 1.53e-1 −0.147 9.27e-4 −0.073 1.01e-1 TopBP1 ENSG00000163781 0.067 1.36e-1 0.051 2.57e-1 −0.020 6.57e-1 Pin1 ENSG00000127445 0.250 1.40e-8 0.571 9.56e-45 0.187 2.52e-5 MRE11 ENSG00000020922 0.063 1.56e-1 −0.007 8.81e-1 −0.024 5.93e-1 PML ENSG00000140464 0.072 1.05e-1 0.217 9.36e-7 0.166 1.85e-4 p63 ENSG00000073282 −0.120 7.04e-3 −0.283 1.08e-10 −0.198 7.71e-6 p73 ENSG00000078900 0.104 2.03e-2 0.258 4.67e-9 0.097 3.02e-2 Supplementary Table S2. -
Incision of Damaged DNA in the Presence of an Impaired Smc5/6 Complex Imperils Genome Stability Jie Peng and Wenyi Feng*
10216–10229 Nucleic Acids Research, 2016, Vol. 44, No. 21 Published online 17 August 2016 doi: 10.1093/nar/gkw720 Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability Jie Peng and Wenyi Feng* Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA Received February 01, 2016; Revised August 05, 2016; Accepted August 08, 2016 ABSTRACT cleotide excision repair (RER) pathways, respectively (7). / Virtually all DNA repair pathways require recognition and The Smc5 6 complex is implicated in homologous subsequent excisions of the damaged nucleotide, giving rise recombination-mediated DNA repair during DNA to a single-stranded gap in the DNA polymer (7). The mech- damage or replication stress. Here, we analysed anisms ensuring such a structural alteration of the chro- genome-wide replication dynamics in a hypomorphic mosome is efficiently repaired and does not lead to further budding yeast mutant, smc6-P4. The overall replica- degradation are underexplored. Conceivably, these mecha- tion dynamics in the smc6 mutant is similar to that nisms would involve protein complexes directly interacting in the wild-type cells. However, we captured a dif- with the chromosomal DNA and implicated in DNA dam- ference in the replication profile of an early S phase age or replication stress response. sample in the mutant, prompting the hypothesis that One of the three evolutionarily conserved Structure the mutant incorporates ribonucleotides and/or ac- Maintenance of Chromosome (SMC) protein complexes, the Smc5/6 complex, plays a key role in DNA damage re- cumulates single-stranded DNA gaps during replica- pair. -
Working on Genomic Stability: from the S-Phase to Mitosis
G C A T T A C G G C A T genes Review Working on Genomic Stability: From the S-Phase to Mitosis Sara Ovejero 1,2,3,* , Avelino Bueno 1,4 and María P. Sacristán 1,4,* 1 Instituto de Biología Molecular y Celular del Cáncer (IBMCC), Universidad de Salamanca-CSIC, Campus Miguel de Unamuno, 37007 Salamanca, Spain; [email protected] 2 Institute of Human Genetics, CNRS, University of Montpellier, 34000 Montpellier, France 3 Department of Biological Hematology, CHU Montpellier, 34295 Montpellier, France 4 Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain * Correspondence: [email protected] (S.O.); [email protected] (M.P.S.); Tel.: +34-923-294808 (M.P.S.) Received: 31 January 2020; Accepted: 18 February 2020; Published: 20 February 2020 Abstract: Fidelity in chromosome duplication and segregation is indispensable for maintaining genomic stability and the perpetuation of life. Challenges to genome integrity jeopardize cell survival and are at the root of different types of pathologies, such as cancer. The following three main sources of genomic instability exist: DNA damage, replicative stress, and chromosome segregation defects. In response to these challenges, eukaryotic cells have evolved control mechanisms, also known as checkpoint systems, which sense under-replicated or damaged DNA and activate specialized DNA repair machineries. Cells make use of these checkpoints throughout interphase to shield genome integrity before mitosis. Later on, when the cells enter into mitosis, the spindle assembly checkpoint (SAC) is activated and remains active until the chromosomes are properly attached to the spindle apparatus to ensure an equal segregation among daughter cells. -
S41467-017-00634-0.Pdf
ARTICLE DOI: 10.1038/s41467-017-00634-0 OPEN BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination Weiran Feng 1,2 & Maria Jasin1,2 Mutations in the tumor suppressor BRCA2 predominantly predispose to breast cancer. Paradoxically, while loss of BRCA2 promotes tumor formation, it also causes cell lethality, although how lethality is triggered is unclear. Here, we generate BRCA2 conditional non- transformed human mammary epithelial cell lines using CRISPR-Cas9. Cells are inviable upon BRCA2 loss, which leads to replication stress associated with under replication, causing mitotic abnormalities, 53BP1 nuclear body formation in the ensuing G1 phase, and G1 arrest. Unexpected from other systems, the role of BRCA2 in homologous recombination, but not in stalled replication fork protection, is primarily associated with supporting human mammary epithelial cell viability, and, moreover, preventing replication stress, a hallmark of pre- cancerous lesions. Thus, we uncover a DNA under replication-53BP1 nuclear body formation- G1 arrest axis as an unanticipated outcome of homologous recombination deficiency, which triggers cell lethality and, we propose, serves as a barrier that must be overcome for tumor formation. 1 Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. 2 Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Correspondence and requests for materials should be addressed to M.J. (email: [email protected]) NATURE COMMUNICATIONS | 8: 525 | DOI: 10.1038/s41467-017-00634-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00634-0 onoallelic inheritance of a deleterious mutation in Results the BRCA1 or BRCA2 tumor suppressor confers BRCA2 is essential for human mammary MCF10A cell viability. -
Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases A, D, and E in Saccharomyces Cerevisiae
INVESTIGATION Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases a, d, and e in Saccharomyces cerevisiae Marion Dubarry,* Conor Lawless,* A. Peter Banks,† Simon Cockell,‡ and David Lydall*,1 *Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, †High Throughput Screening Facility, Newcastle Biomedicine, and ‡Bioinformatics Support Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom ORCID IDs: 0000-0002-4186-8506 (C.L.); 0000-0003-2478-085X (D.L.) ABSTRACT Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase KEYWORDS a-primase (Pol a) and DNA polymerase d (Pol d) replicate the lagging-strand and Pol a and DNA polymerase e DNA replication (Pol e) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed DNA polymerase genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We Profilyzer combined temperature-sensitive mutations affecting the three replicative polymerases, Pol a,Pold,and DIXY Pol e with genome-wide collections of null and reduced function mutations. We identify large numbers of quantitative genetic interactions that inform about the roles that specific genes play to help Pol a,Pold, and Pol e function. fitness analyses Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not (QFA) represent the majority of the genetic interactions identified. Instead our data support a model for division of Saccharomyces labor between the different DNA polymerases during DNA replication. For example, our genetic interaction cerevisiae data are consistent with biochemical data showing that Pol e is more important to the Pre-Loading complex than either Pol a or Pol d. -
Arsenic Hexoxide Has Differential Effects on Cell Proliferation And
www.nature.com/scientificreports OPEN Arsenic hexoxide has diferential efects on cell proliferation and genome‑wide gene expression in human primary mammary epithelial and MCF7 cells Donguk Kim1,7, Na Yeon Park2,7, Keunsoo Kang3, Stuart K. Calderwood4, Dong‑Hyung Cho2, Ill Ju Bae5* & Heeyoun Bunch1,6* Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer efects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA‑sequencing and bioinformatics analyses, diferentially expressed genes in signifcantly afected biological pathways in these cell types were validated by real‑time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly diferential efects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without afecting the growth of HUMEC in a dose‑dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are signifcantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6‑treated MCF7 cells.