Mcm10 Has Potent Strand-Annealing Activity and Limits Translocase-Mediated Fork Regression
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A Mutation in DNA Polymerase Α Rescues WEE1KO Sensitivity to HU
International Journal of Molecular Sciences Article A Mutation in DNA Polymerase α Rescues WEE1KO Sensitivity to HU Thomas Eekhout 1,2 , José Antonio Pedroza-Garcia 1,2 , Pooneh Kalhorzadeh 1,2, Geert De Jaeger 1,2 and Lieven De Veylder 1,2,* 1 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium; [email protected] (T.E.); [email protected] (J.A.P.-G.); [email protected] (P.K.); [email protected] (G.D.J.) 2 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium * Correspondence: [email protected] Abstract: During DNA replication, the WEE1 kinase is responsible for safeguarding genomic integrity by phosphorylating and thus inhibiting cyclin-dependent kinases (CDKs), which are the driving force of the cell cycle. Consequentially, wee1 mutant plants fail to respond properly to problems arising during DNA replication and are hypersensitive to replication stress. Here, we report the identification of the pola-2 mutant, mutated in the catalytic subunit of DNA polymerase α, as a suppressor mutant of wee1. The mutated protein appears to be less stable, causing a loss of interaction with its subunits and resulting in a prolonged S-phase. Keywords: replication stress; DNA damage; cell cycle checkpoint Citation: Eekhout, T.; Pedroza- 1. Introduction Garcia, J.A.; Kalhorzadeh, P.; De Jaeger, G.; De Veylder, L. A Mutation DNA replication is a highly complex process that ensures the chromosomes are in DNA Polymerase α Rescues correctly replicated to be passed onto the daughter cells during mitosis. Replication starts WEE1KO Sensitivity to HU. Int. -
Saccharomyces Rrm3p, a 5 to 3 DNA Helicase That Promotes Replication
Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Saccharomyces Rrm3p, a 5 to 3 DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA Andreas S. Ivessa,1 Jin-Qiu Zhou,1,2 Vince P. Schulz, Ellen K. Monson, and Virginia A. Zakian3 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric C1–3A/TG1–3 tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5 ,to 3 DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere at the natural X-bearing Chromosome III-L telomere, and at Y-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C1–3A/TG1–3 DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication. [Key Words: Telomere; helicase; telomerase; replication; RRM3; yeast] Received February 7, 2002; revised version accepted April 10, 2002. Telomeres are the natural ends of eukaryotic chromo- Because conventional DNA polymerases cannot repli- somes. In most organisms, the very ends of chromo- cate the very ends of linear DNA molecules, special somes consist of simple repeated sequences. For ex- mechanisms are required to prevent the loss of terminal ample, Saccharomyces cerevisiae chromosomes end in DNA. -
The Architecture of a Eukaryotic Replisome
The Architecture of a Eukaryotic Replisome Jingchuan Sun1,2, Yi Shi3, Roxana E. Georgescu3,4, Zuanning Yuan1,2, Brian T. Chait3, Huilin Li*1,2, Michael E. O’Donnell*3,4 1 Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA 2 Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York, USA. 3 The Rockefeller University, 1230 York Avenue, New York, New York, USA. 4 Howard Hughes Medical Institute *Correspondence and requests for materials should be addressed to M.O.D. ([email protected]) or H.L. ([email protected]) ABSTRACT At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase leads the way in front to unwind DNA, and that DNA polymerases (Pol) trail behind the helicase. Here we use single particle electron microscopy to directly image a replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, while Ctf4 and the lagging strand Pol α-primase (Pol α) are behind the helicase. This unexpected architecture indicates that the leading strand DNA travels a long distance before reaching Pol ε, it first threads through the Mcm2-7 ring, then makes a U-turn at the bottom to reach Pol ε at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture, and provides a basis for further structural and biochemical replisome studies. INTRODUCTION DNA is replicated by a multi-protein machinery referred to as a replisome 1,2. Replisomes contain a helicase to unwind DNA, DNA polymerases that synthesize the leading and lagging strands, and a primase that makes short primed sites to initiate DNA synthesis on both strands. -
Supporting Information
Supporting Information Figure S1. The functionality of the tagged Arp6 and Swr1 was confirmed by monitoring cell growth and sensitivity to hydeoxyurea (HU). Five-fold serial dilutions of each strain were plated on YPD with or without 50 mM HU and incubated at 30°C or 37°C for 3 days. Figure S2. Localization of Arp6 and Swr1 on chromosome 3. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 3L, Tel 3R, CEN3, and the RP gene are shown under the panels. Figure S3. Localization of Arp6 and Swr1 on chromosome 4. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) in the whole chromosome region are compared. The position of Tel 4L, Tel 4R, CEN4, SWR1, and RP genes are shown under the panels. Figure S4. Localization of Arp6 and Swr1 on the region including the SWR1 gene of chromosome 4. The binding of Arp6- FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position and orientation of the SWR1 gene is shown. Figure S5. Localization of Arp6 and Swr1 on chromosome 5. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 5L, Tel 5R, CEN5, and the RP genes are shown under the panels. Figure S6. Preferential localization of Arp6 and Swr1 in the 5′ end of genes. Vertical bars represent the binding ratio of proteins in each locus. -
The Cdc23 (Mcm10) Protein Is Required for the Phosphorylation of Minichromosome Maintenance Complex by the Dfp1-Hsk1 Kinase
The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase Joon-Kyu Lee*†, Yeon-Soo Seo†, and Jerard Hurwitz*‡ *Program in Molecular Biology, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; and †Department of Biological Science, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea Contributed by Jerard Hurwitz, December 4, 2002 Previous studies in Saccharomyces cerevisiae have defined an (9). Furthermore, studies in S. cerevisiae with mcm degron essential role for the Dbf4-Cdc7 kinase complex in the initiation of mutants showed that the Mcm proteins are also required for the DNA replication presumably by phosphorylation of target proteins, progression of the replication fork (10). These observations such as the minichromosome maintenance (Mcm) complex. We suggest that the Mcm complex is the replicative helicase. have examined the phosphorylation of the Mcm complex by the Cdc7, a serine͞threonine kinase conserved from yeast to Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of humans (reviewed in ref. 11), is activated by the regulatory Dbf4-Cdc7. In vitro, the purified Dfp1-Hsk1 kinase efficiently phos- protein Dbf4. Although the level of Cdc7p is constant through- ͞ phorylated Mcm2p. In contrast, Mcm2p, present in the six-subunit out the cell cycle, the activity of this kinase peaks at the G1 S Mcm complex, was a poor substrate of this kinase and required transition, concomitant with the cellular level of Dbf4p. In S. ͞ Cdc23p (homologue of Mcm10p) for efficient phosphorylation. In cerevisiae, Dbf4p binds to chromatin at the G1 S transition and the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and remains attached to chromatin during S phase (12). -
The Biochemical Activities of the Saccharomyces Cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain
G C A T T A C G G C A T genes Article The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain David G. Nickens y, Christopher W. Sausen y and Matthew L. Bochman * Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN 47405, USA; [email protected] (D.G.N.); [email protected] (C.W.S.) * Correspondence: [email protected] These authors contributed equally to this work. y Received: 31 March 2019; Accepted: 20 May 2019; Published: 28 May 2019 Abstract: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1DN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1DN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity. Keywords: DNA helicase; Saccharomyces cerevisiae; Pif1; telomerase; telomere 1. Introduction DNA helicases are enzymes that couple DNA binding and ATP hydrolysis to unwind double-stranded DNA (dsDNA) into its component single strands [1]. -
Exploring the Non-Canonical Functions of Metabolic Enzymes Peiwei Huangyang1,2 and M
© 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm033365. doi:10.1242/dmm.033365 REVIEW SPECIAL COLLECTION: CANCER METABOLISM Hidden features: exploring the non-canonical functions of metabolic enzymes Peiwei Huangyang1,2 and M. Celeste Simon1,3,* ABSTRACT A key finding from studies of metabolic enzymes is the existence The study of cellular metabolism has been rigorously revisited over the of mechanistic links between their nuclear localization and the past decade, especially in the field of cancer research, revealing new regulation of transcription. By modulating gene expression, insights that expand our understanding of malignancy. Among these metabolic enzymes themselves facilitate adaptation to rapidly insights isthe discovery that various metabolic enzymes have surprising changing environments. Furthermore, they can directly shape a ’ activities outside of their established metabolic roles, including in cell s epigenetic landscape (Kaelin and McKnight, 2013). the regulation of gene expression, DNA damage repair, cell cycle Strikingly, several metabolic enzymes exert completely distinct progression and apoptosis. Many of these newly identified functions are functions in different cellular compartments. Nuclear fructose activated in response to growth factor signaling, nutrient and oxygen bisphosphate aldolase, for example, directly interacts with RNA ́ availability, and external stress. As such, multifaceted enzymes directly polymerase III to control transcription (Ciesla et al., 2014), -
Assembly of the Cdc45-Mcm2–7-GINS Complex in Human Cells Requires the Ctf4/And-1, Recql4, and Mcm10 Proteins
Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins Jun-Sub Ima,1, Sang-Hee Kia,1, Andrea Farinab, Dong-Soo Junga, Jerard Hurwitzb,2, and Joon-Kyu Leea,2 aDepartment of Biology Education, Seoul National University, Seoul, 151-748, Korea; and bProgram in Molecular Biology, Memorial Sloan–Kettering Cancer Center, New York, NY 10021 Contributed by Jerard Hurwitz, July 17, 2009 (sent for review July 1, 2009) In eukaryotes, the activation of the prereplicative complex and Cdc45, and GINS (the CMG complex), purified from Drosophila assembly of an active DNA unwinding complex are critical but embryos, has DNA helicase activity in vitro (12). poorly understood steps required for the initiation of DNA repli- In addition to the above components, the assembly of the DNA cation. In this report, we have used bimolecular fluorescence unwinding complex also depends on additional factors including complementation assays in HeLa cells to examine the interactions Dpb11/Cut5, Sld2, Sld3, and Mcm10. Mcm10 is required for the between Cdc45, Mcm2–7, and the GINS complex (collectively called chromatin binding of Cdc45 and DNA polymerase ␣ in yeast and the CMG complex), which seem to play a key role in the formation Xenopus (13–16). Dpb11/Cut5 is essential for origin binding of and progression of replication forks. Interactions between the GINS in Saccharomyces cerevisiae (17), and reported to be required CMG components were observed only after the G1/S transition of for chromatin binding of Cdc45 and DNA polymerase ␣ in Xenopus the cell cycle and were abolished by treatment of cells with either (18). -
Huh7 HK4+ HK2- Cells a Protein Complementation Assay B Coimmunoprecipitation Even If NS3 Is Able to Stimulates Glycolysis in Cells Expressing Replicate
Dengue virus protein NS3 activates hexokinase activity in SAT-390 hepatocytes to support virus replication Marianne FIGL, Clémence JACQUEMIN, Patrice ANDRE, Laure PERRIN-COCON, Vincent LOTTEAU, Olivier DIAZ International Center for Infectiology Research (CIRI), INSERM U1111, CNRS UMR5308, Université de Lyon, FRANCE 1 INTRODUCTION 4 RESULTS 5 CONCLUSIONS Result 2: DENV NS3 protein interacts with hexokinases Viruses are mandatory parasites that use metabolism machinery to Result 1: DENV efficiently replicates in HuH7 HK4+ HK2- cells A Protein Complementation Assay B Coimmunoprecipitation Even if NS3 is able to stimulates glycolysis in cells expressing replicate. Growing literature demonstrates that viruses manipulate A.A. DENV-NS3 versus human metabolism enzymes DENVB.B.-NS3 versus hexokinases A. HuH7 HuH7 HK4+ HK2- B. (a) (b) 60 Lysate Co-IP HK2 or HK4, we observe an higher DENV replication in HuH7 central carbon metabolism (CCM) and more specifically glycolysis for HuH7 HuH7 55 NS3-3xFlag - + - + HuH7 HK4+ HK2- HK4+ HK2- suggesting that HK4 positive cells are more susceptible their propagation [1]. However, the underlying mechanisms are not HuH7 HK4+ HK2- 50 HK1 α-Gluc 45 α-Flag to DENV replication. fully described. Our team has already demonstrated that hepatitis C 40 80 80 HK2 α-Gluc *** 35 NS5A protein interacts and activates hexokinases (HKs) to favor viral 70 70 α-Flag cells 30 Poster presented at: presented Poster Fluorescente light Fluorescente 60 cells 60 α-Gluc replication [2]. It was described that dengue infection (DENV) 25 HK3 We observed that HuH7 HK4+HK2- cells have a rewiring of their 50 50 α-Flag 20 increases glycolysis [3] and thus we wondered if control of 40 40 glycolytic pathway resulting in intracellular lipids accumulation (see 15 HK4 α-Gluc 30 hexokinase activity was shared by DENV, another Flavivirus. -
Is Glyceraldehyde-3-Phosphate Dehydrogenase a Central Redox Mediator?
1 Is glyceraldehyde-3-phosphate dehydrogenase a central redox mediator? 2 Grace Russell, David Veal, John T. Hancock* 3 Department of Applied Sciences, University of the West of England, Bristol, 4 UK. 5 *Correspondence: 6 Prof. John T. Hancock 7 Faculty of Health and Applied Sciences, 8 University of the West of England, Bristol, BS16 1QY, UK. 9 [email protected] 10 11 SHORT TITLE | Redox and GAPDH 12 13 ABSTRACT 14 D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an immensely important 15 enzyme carrying out a vital step in glycolysis and is found in all living organisms. 16 Although there are several isoforms identified in many species, it is now recognized 17 that cytosolic GAPDH has numerous moonlighting roles and is found in a variety of 18 intracellular locations, but also is associated with external membranes and the 19 extracellular environment. The switch of GAPDH function, from what would be 20 considered as its main metabolic role, to its alternate activities, is often under the 21 influence of redox active compounds. Reactive oxygen species (ROS), such as 22 hydrogen peroxide, along with reactive nitrogen species (RNS), such as nitric oxide, 23 are produced by a variety of mechanisms in cells, including from metabolic 24 processes, with their accumulation in cells being dramatically increased under stress 25 conditions. Overall, such reactive compounds contribute to the redox signaling of the 26 cell. Commonly redox signaling leads to post-translational modification of proteins, 27 often on the thiol groups of cysteine residues. In GAPDH the active site cysteine can 28 be modified in a variety of ways, but of pertinence, can be altered by both ROS and 29 RNS, as well as hydrogen sulfide and glutathione. -
Additional File 4. P. Vinckei Genes Ordered According to the Gene Expression of Their Orthologs in P
A fast and cost-effective microsampling protocol incorporating reduced animal usage for time- series transcriptomics in rodent malaria parasites Item Type Article Authors Ramaprasad, Abhinay; Subudhi, Amit; Culleton, Richard; Pain, Arnab Citation Ramaprasad A, Subudhi AK, Culleton R, Pain A (2019) A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites. Malaria Journal 18. Available: http:// dx.doi.org/10.1186/s12936-019-2659-4. Eprint version Publisher's Version/PDF DOI 10.1186/s12936-019-2659-4 Publisher Springer Nature Journal Malaria Journal Rights This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Download date 28/09/2021 01:24:39 Item License http://creativecommons.org/licenses/by/4.0/ Link to Item http://hdl.handle.net/10754/628424 Additional file 4. P. vinckei genes ordered according to the gene expression of their orthologs in P. falciparum. Pfalciparum ortholog gene_id 6h 12h 18h 24h Product PF3D7_1432500 PVVCY_1001720 6.742 -
Mcm10 and And-1/CTF4 Recruit DNA Polymerase to Chromatin For
Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mcm10 and And-1/CTF4 recruit DNA polymerase ␣ to chromatin for initiation of DNA replication Wenge Zhu,1,3 Chinweike Ukomadu,1,3 Sudhakar Jha,1,3 Takeshi Senga,1 Suman K. Dhar,1 James A. Wohlschlegel,1 Leta K. Nutt,2 Sally Kornbluth,2 and Anindya Dutta1,4 1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA; 2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase–polymerase complex, DNA polymerase ␣ (pol ␣), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol ␣. And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol ␣, and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10–And-1 interaction interferes with the loading of And-1 and of pol ␣, inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol ␣–primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery.