Additional File 4. P. Vinckei Genes Ordered According to the Gene Expression of Their Orthologs in P
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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. -
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. -
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), -
Mcm10 Has Potent Strand-Annealing Activity and Limits Translocase-Mediated Fork Regression
Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression Ryan Maylea, Lance Langstona,b, Kelly R. Molloyc, Dan Zhanga, Brian T. Chaitc,1,2, and Michael E. O’Donnella,b,1,2 aLaboratory of DNA Replication, The Rockefeller University, New York, NY 10065; bHoward Hughes Medical Institute, The Rockefeller University, New York, NY 10065; and cLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065 Contributed by Michael E. O’Donnell, November 19, 2018 (sent for review November 8, 2018; reviewed by Zvi Kelman and R. Stephen Lloyd) The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, of function using genetics, cell biology, and cell extracts have GINS) tightly binds Mcm10, an essential replication protein in all identified Mcm10 functions in replisome stability, fork progres- eukaryotes. Here we show that Mcm10 has a potent strand- sion, and DNA repair (21–25). Despite significant advances in the annealing activity both alone and in complex with CMG. CMG- understanding of Mcm10’s functions, mechanistic in vitro studies Mcm10 unwinds and then reanneals single strands soon after they of Mcm10 in replisome and repair reactions are lacking. have been unwound in vitro. Given the DNA damage and replisome The present study demonstrates that Mcm10 on its own rap- instability associated with loss of Mcm10 function, we examined the idly anneals cDNA strands even in the presence of the single- effect of Mcm10 on fork regression. Fork regression requires the strand (ss) DNA-binding protein RPA, a property previously unwinding and pairing of newly synthesized strands, performed by associated with the recombination protein Rad52 (26). -
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. -
Regulating Telomerase Access to the Telomere
Journal of Cell Science 113, 3357-3364 (2000) 3357 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1074 COMMENTARY Positive and negative regulation of telomerase access to the telomere Sara K. Evans and Victoria Lundblad* Department of Molecular and Human Genetics, and Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 USA *Author for correspondence (e-mail: [email protected]) Published on WWW 13 September 2000 SUMMARY The protective caps on chromosome ends – known as to be achieved through recruitment of the enzyme by the telomeres – consist of DNA and associated proteins that are end-binding protein Cdc13p. In contrast, duplex-DNA- essential for chromosome integrity. A fundamental part of binding proteins assembled along the telomeric tract exert ensuring proper telomere function is maintaining adequate a feedback system that negatively modulates telomere length of the telomeric DNA tract. Telomeric repeat length by limiting the action of telomerase. In mammalian sequences are synthesized by the telomerase reverse cells, and perhaps also in yeast, binding of these proteins transcriptase, and, as such, telomerase is a central player probably promotes a higher-order structure that renders in the maintenance of steady-state telomere length. the telomere inaccessible to the telomerase enzyme. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively Key words: Telomere, Telomerase access, Positive length regulation, control access of telomerase to the chromosome terminus. Negative length regulation, Cdc13, Lagging strand synthesis, Rap1, In yeast, positive regulation of telomerase access appears TRF1, TRF2 INTRODUCTION processing activities such as active degradation or deletion events. -
DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer
cancers Review DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer Youri I. Pavlov 1,2,* , Anna S. Zhuk 3 and Elena I. Stepchenkova 2,4 1 Eppley Institute for Research in Cancer and Allied Diseases and Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 2 Department of Genetics and Biotechnology, Saint-Petersburg State University, 199034 Saint Petersburg, Russia; [email protected] 3 International Laboratory of Computer Technologies, ITMO University, 197101 Saint Petersburg, Russia; [email protected] 4 Laboratory of Mutagenesis and Genetic Toxicology, Vavilov Institute of General Genetics, Saint-Petersburg Branch, Russian Academy of Sciences, 199034 Saint Petersburg, Russia * Correspondence: [email protected] Received: 30 September 2020; Accepted: 13 November 2020; Published: 24 November 2020 Simple Summary: The etiology of cancer is linked to the occurrence of mutations during the reduplication of genetic material. Mutations leading to low replication fidelity are the culprits of many hereditary and sporadic cancers. The archetype of the current model of replication fork was proposed 30 years ago. In the sequel to our 2010 review with the words “years after” in the title inspired by A. Dumas’s novels, we go over new developments in the DNA replication field and analyze how they help elucidate the effects of the genetic variants of DNA polymerases on cancer. Abstract: Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. -
Profoldin Human DNA Polymerase Alpha Assay Kits
ProFoldin 10 Technology Drive, Suite 40, Number 188 Hudson, MA 01749-2791 USA Tel: (508)-735-2539 FAX: (508) 845-9258 www.profoldin.com [email protected] INSTRUCTIONS ProFoldin Human DNA Polymerase Alpha Assay Kits Human DNA Polymerase Alpha Assay Kit Catalog No: HDPA100K Human DNA Polymerase Alpha Assay Kit Plus Catalog No: HDPA100KE Introduction Human DNA Polymerase Alpha (Pol α) is responsible for the initiation of DNA replication at origins of replication The Pol α enzyme consists of four subunits: the catalytic subunit POLA1, the regulatory subunit POLA2, and the small and the large primase subunits PRIM1 and PRIM2. The human DNA polymerase alpha assay is based on measurement of the DNA molecules synthesized by the DNA polymerase. The assay is performed in a 384-well or 96-well plate format. The assay can be used for detection of DNA polymerase alpha activity and high throughput screen of human DNA polymerase alpha inhibitors. The Human DNA Polymerase Alpha Assay Kit (Catalog No. HDPA100K) includes all the assay kit components except the enzyme for 100 assays in a 384-well plate assay format: 400 µl of 10 x Buffer, 33 µl of 100 x DNA template, 33 µl of 100 x dNTP mix, 1550 µl of 2 x Dye, 1550 µl of 50 mM EDTA. The kit does not include the enzyme. The Human DNA Polymerase Alpha Assay Kit Plus (Catalog No. DPG100KE) includes all the assay kit components for 100 assays in 384-well plate assay format: 400 µl of 10 x Buffer, 33 µl of 100 x DNA template, 33 µl of 100 x dNTP mix, 33 µl of 100 x human DNA polymerase alpha, 1550 µl of 2 x Dye, 1550 µl of 50 mM EDTA. -
Open Full Page
Published OnlineFirst December 17, 2014; DOI: 10.1158/1541-7786.MCR-14-0381 Cell Cycle and Senescence Molecular Cancer Research DNA-Directed Polymerase Subunits Play a Vital Role in Human Telomeric Overhang Processing Raffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, and Diego Loayza Abstract Telomeres consist of TTAGGG repeats bound by the shelterin could also show that the CST, shelterin, and polymerase com- complex and end with a 30 overhang. In humans, telomeres plexes interact, revealing contacts occurring at telomeres. We shorten at each cell division, unless telomerase (TERT) is found that the polymerase complex could associate with telome- expressed and able to add telomeric repeats. For effective telomere rase activity. Finally, depletion of p180 by siRNA led to increased maintenance, the DNA strand complementary to that made by overhang amounts at telomeres. These data support a model in telomerase must be synthesized. Recent studies have discovered a which the polymerase complex is important for proper telomeric link between different activities necessary to process telomeres overhang processing through fill-in synthesis, during S phase. in the S phase of the cell cycle to reform a proper overhang. These results shed light on important events necessary for efficient Notably, the human CST complex (CTC1/STN1/TEN1), known to telomere maintenance and protection. interact functionally with the polymerase complex (POLA/pri- mase), was shown to be important for telomere processing. Here, Implications: This study describes the interplay between DNA focus was paid to the catalytic (POLA1/p180) and accessory replication components with proteins that associate with chro- (POLA2/p68) subunits of the polymerase, and their mechanistic mosome ends, and telomerase. -
Profoldin Human DNA Polymerase Alpha
ProFoldin 10 Technology Drive, Suite 40, Number 188 Hudson, MA 01749-2791 USA Tel: (508)-735-2539 FAX: (508) 845-9258 www.profoldin.com [email protected] INSTRUCTIONS ProFoldin Human DNA Polymerase Alpha Human DNA Polymerase Alpha – for 100 assays Catalog No: HDPA1-100 Human DNA Polymerase Alpha – for 1000 assays Catalog No: HDPA1-1000 Protein construct: Wild-type human DNA polymerase alpha catalytic subunit purified from a baculovirus insect protein expression system. MW: 170 kDa Enzyme concentration: 4 µM DNA polymerase assay: The DNA polymerase beta activity is measured by using the Human DNA Polymerase Assay Alpha kit (Catalog No. HDPA100K). Storage temperature: -20 or -80C. Do not freeze-and-thaw repeatedly. Enzyme dilution: Use the 1 x assay to dilute the enzyme just before the assay. Do not store diluted enzyme solution. Human DNA Polymerase Alpha – for 100 assays (Catalog No: HDPA1-100 ) includes 7 l of 4 µM human DNA polymerase alpha catalytic subunit. It is sufficient for 100 assays. Human DNA Polymerase Alpha – for 1000 assays (Catalog No: HDPA1-1000) includes 50 l of 4 µM human DNA polymerase alpha catalytic subunit. It is sufficient for 1000 assays. Assay Protocol using Human DNA Polymerase Alpha Assay kit (Catalog No. HDPA100K) The following assay protocol is based on the 384-well plate assay format (plate type: Matrix 4318 or alike). The reaction volume is 30 l and the final assay volume is 60 l. For 96-well plate assays (plate type: Costar 3915 or alike), the reaction volume is 60 l and the final assay volume is 120 l. -
NAPRALERT Classification Codes
NAPRALERT Classification Codes June 1993 STN International® Copyright © 1993 American Chemical Society Quoting or copying of material from this publication for educational purposes is encouraged, providing acknowledgement is made of the source of such material. Classification Codes in NAPRALERT The NAPRALERT File contains classification codes that designate pharmacological activities. The code and a corresponding textual description are searchable in the /CC field. To be comprehensive, both the code and the text should be searched. Either may be posted, but not both. The following tables list the code and the text for the various categories. The first two digits of the code describe the categories. Each table lists the category described by codes. The last table (starting on page 56) lists the Classification Codes alphabetically. The text is followed by the code that also describes the category. General types of pharmacological activities may encompass several different categories of effect. You may want to search several classification codes, depending upon how general or specific you want the retrievals to be. By reading through the list, you may find several categories related to the information of interest to you. For example, if you are looking for information on diabetes, you might want to included both HYPOGLYCEMIC ACTIVITY/CC and ANTIHYPERGLYCEMIC ACTIVITY/CC and their codes in the search profile. Use the EXPAND command to verify search terms. => S HYPOGLYCEMIC ACTIVITY/CC OR 17006/CC OR ANTIHYPERGLYCEMIC ACTIVITY/CC OR 17007/CC 490 “HYPOGLYCEMIC”/CC 26131 “ACTIVITY”/CC 490 HYPOGLYCEMIC ACTIVITY/CC ((“HYPOGLYCEMIC”(S)”ACTIVITY”)/CC) 6 17006/CC 776 “ANTIHYPERGLYCEMIC”/CC 26131 “ACTIVITY”/CC 776 ANTIHYPERGLYCEMIC ACTIVITY/CC ((“ANTIHYPERGLYCEMIC”(S)”ACTIVITY”)/CC) 3 17007/CC L1 1038 HYPOGLYCEMIC ACTIVITY/CC OR 17006/CC OR ANTIHYPERGLYCEMIC ACTIVITY/CC OR 17007/CC 2 This search retrieves records with the searched classification codes such as the ones shown here. -
Characterizing the 3D Interactions of the Base-Pair Stems
CHARACTERIZING THE 3D INTERACTIONS OF THE BASE-PAIR STEMS AND HELICES IN RNA MOLECULES BY ZAINAB MOHAMMED HASSAN FADHIL A thesis submitted to the The Graduate School- New Brunswick Rutgers, the state University of New Jersey In partial fulfillment of the requirements For the degree of Master of Science Graduate Program in Chemistry and Chemical Biology Written under the direction of Dr. Wilma K. Olson And approved By ………………………………… ………………………………… ………………………………… New Brunswick, New Jersey October 2017 ABSTRACT OF THE THESIS CHARACTERIZING THE 3D INTERACTIONS OF THE BASE-PAIR STEMS AND HELICES IN RNA MOLECULES By ZAINAB MOHAMMED HASSAN FADHIL Thesis Director: Wilma K. Olson Characterizing RNA structure experimentally is a challenging task. Computational methods complement experimental methods. In this thesis, we study, using software tools such as DSSR, Matlab, and Excel, the three-dimensional structures of a group of non-redundant RNA structures. These structures are those identifiers as Leontis and Zirbel at January 2017 [1], and the relevant coordinates for these structures downloaded from the Protein Data Bank (PDB) [2]. Specifically, we find the angles between all pairs of chemically linked double helical stems in each of the RNA structures, the distances between the mid points between all these stems, and the distributions for the calculated angles and distances. Additionally, we separate the above calculations to consider stems within the same helix. A helix is composed of at least two stacked base pairs and these base pairs are not necessarily chemically linked. The third part contains these calculations separated for stems in different helices. Based on these calculations, the stems in the same helix can be classified into two groups.