DOCSLIB.ORG

Interaction of Escherichia Coli Dnab and Dnac(D) Gene Products in Vitro

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interaction of Escherichia Coli Dnab and Dnac(D) Gene Products in Vitro Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 921-925, March 1975 Interaction of Escherichia coli dnaB and dnaC(D) Gene Products In Vitro (DNA replication/DNA-stimulated ATPase) SUE WICKNER* AND JERARD HURWITZt * Viral Carcinogenesis Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20014; and t Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York, 10461 Contributed by Jerard Hurwitz, December 24, 1974 ABSTRACT Purified E. coli dnaB and dnaC(D) gene tation in the 4X174 DNA-synthesizing system. The assay products interact physically and functionally in vitro. This interaction was demonstrated as follows: (a) A com- conditions, definitions of units, and purification procedures plex of dnaB and dnaC(D) gene products was isolated by were described previously for dnaB (19, 21) and dnaC(D) gel filtration; ATP specifically was required for isolation gene products (19, 16). The dnaB gene product contained no of the complex. (b) The DNA-independent ribonucleoside detectable dnaC(D) complementing activity (<5 X 10-4 triphosphatase activity associated with dnaB gene product U/0.4 U of dnaB), and dnaC(D) gene product contained no was inhibited by dnaC(D) gene product. (c) The dnaC(D) gene product was protected from inactivation by N-ethyl- detectable dnaB activity [<5 X 10-4 U/0.5 U of dnaC(D)]. maleimide by the combination of dnaB gene product and The addition of dnaB (0.12 U) to the dnaC(D) comp)lementa- ATP; this protection required ATP specifically. tion assay system with dnaC(D) (0.015 U) did not affect the dnaC(D) complementing activity; the addition of dnaC(D) Some of the proteins involved in Escherichia coli DNA rep- (0.15 U) to the dnaB complementation assay system with lication have been defined by temperature-sensitive mutants dnaB (0.012 U) did not affect the dnaB complementing ac- that do not replicate their DNA at elevated temperatures. tivity. The genes that determine these proteins have been designated dnaA (1), dnaB (2), dnaC(D) (3), dnaE (4), dnaG (3), dnafH Assay for A TPase. Reaction mixtures (0.03 ml) contained (5), dnaH (6), dnaI (7), dnaP (8), lig (9), and polA (10). 50 mM Tris HClI, pH 7.5, 1 mM dithiothreitol, 0.4 mM Mutants in dnaE contain thermolabile DNA polymerase III MgCl2, 0.05 mg/ml of bovine serum albumin, 0.2 mM (11, 12); lig mutants contain thermolabile DNA ligase (9, 13, ['y-32P]ATP (50 cpm/pmol), and protein fractions as in- 14); and polA mutants are temperature-sensitive for growth dicated. After 30 min at 30°, Pi was determined by the method when they contain thermolabile 5' to 3' exonuclease (exo- of Conway and Lipmann (22). One unit (U) of ATPase nuclease VI) (15). Of the other proteins known to be involved catalyzed the production of 1 ,umol of 32Pi using the above in E. coli DNA replication, dnaB, C(D), and G have been iso- conditions. lated using in vitro complementation assays in which protein RESULTS preparations from wild-type cells stimulate 4X174 DNA- dependent DNA synthesis in heat-inactivated crude extracts Physical Association of dnaB and dnaC(D) Gene Products. of dna temperature-sensitive cells (16-19). Each of these dna The dnaC(D) gene product was detected (as measured by gene products has been isolated from both wild-type and stimulation in the 4X174 DNA-dependent complementation temperature-sensitive strains and the thermolability of the assay) physically associated with the dnaB gene product (also temperature-sensitive protein has been demonstrated in the measured by the OX174 DNA-dependent complementation 4X174 DNA-dependent complementation system. As yet, no system) when the two purified proteins were mixed and sub- enzymatic activity has been found associated with dnaG or jected to gel filtration through BioGel A-5m agarose in the dnaC(D) gene products. The dnaC and dnaD gene products presence of ATP (Fig. 1A). Incubation of dnaC(D) with dnaB have been shown to be identical in vitro (16) and in vivo (20); gene product prior to gel filtration was not essential and did the protein will be referred to here as dnaC(D) gene product. not result in an increased amount of dnaC(D) associated with Purified dnaB gene product contains ribonucleoside triphos- dnaB. Fig. 1B shows that in the presence of ATP and in the phatase activity that is stimulated by single-stranded DNA. absence of dnaB gene product, dnaC(D) eluted as expected These triphosphatase activities and dnaB complementing for a protein with a molecular weight of about 25,000 (16). activity copurify over the last 20-fold of a 40,000-fold puri- Fig. 1C shows that dnaB gene product in the presence of fication procedure (21). The purpose of this report is to de- ATP and absence of dnaC(D) gene product eluted as expected scribe the physical and functional interaction of two of these for a protein with a molecular weight of about 250,000 (18) $. proteins involved in replication, dnaB and dnaC(D) gene pro- It eluted broadly, suggesting, as shown before (21), that dnaB ducts. gene product can exist in multiple forms that are active in the MATERIALS AND METHODS t Similar results to those shown in Fig. lA and B were obtained Materials, Reagents, and Methods, unless otherwise indi- when columns were developed with 50 mM Tris-HCl (pH 7.5) cated, were as described (16-19). and 50 mM KCl in place of 30 mM potassium phosphate (pH 6.0). However, at pH 7.5, dnaB complementing activity could Isolation of dnaB and dnaC(D) Gene Products. dnaB and not be recovered in the absence of ATP in the presence or absence dnaC(D) gene products were assayed by in vitro complemen- of dnaC(D) gene product. 921 Downloaded by guest on October 1, 2021 922 Biochemistry: Wickner and Hurwitz Proc. Nat. Acad. Sci. USA 72 (1975) DNA-dependent complementation assay. Since the A. dnaB + dnaC gene products + ATP Included OX174 gene product was not significantly 0.16 void volume voume 0.16 elution profile of dnaB affected by its interaction with dnaC(D) gene product, proba- 0.08 0.08 bly only one or a few dnaC(D) molecules are associated with one native dnaB molecule. were the B. dnaC gene product + ATP When dnaB and dnaC(D) gene products mixed in 0.24 absence of ATP and filtered through a column in the absence of ATP, the two proteins were not associated with each other 0.16 (Fig. 1D). Furthermore, most of the dnaB activity was ex- cluded from the column; the nature of this aggregation has 0.08 not been studied, nor has it been demonstrated that it was catalyzed exclusively by the dnaC(D) gene product. In the 0.16 C. dnaB gene product + ATP absence of ATP, dnaB and dnaC(D) gene products alone eluted as they had in the presence of ATP (results similar to 0.08 those shown in Fig. 1B and C). Fig. 1E shows that dnaB and dnaC(D) activities were not associated with each other if they cl D. dnaB + dnaC gene products-ATP were incubated together for 30 min at 300 in the presence of E 024 0.24 ATP and then filtered through the agarose column in the 0.16 0.16 absence of ATP. Thus, ATP is required for detecting the physical complex of dnaB and dnaC(D) gene products. 0.08 0.08 Under conditions as shown in Fig. 1A but with 0, 1, 5, 50, and 11 500 AM ATP, 0, 17, 33, 48, and 52% of the dnaC(D) activity, E. dnaB + dnaC gene products incubated+ATP, respectively, was associated with the dnaB gene product. gel filtration-ATP The nucleotide requirement was specific for ATP. Under the conditions described in Fig. 1A, no dnaC(D) activity was 024 detected associated with dnaB gene product when ATP was 0.16 0.16 replaced with 10AM dATP, dGTP, dTTP, UTP, CTP, GTP, Bry-methylene-ATP, or ADP (<5% of that associated with 0.08 0.08 dnaB gene product in the presence of ATP). The fate of ATP in this reaction is unknown. No detectable 91 _ 0 AMP was produced by dnaB, dnaC(D), or the combination 024 F. 1X dnaB + '/2X dnaC + ATP of gene products. No detectable ['y-'2P]ATP, [a-'2P]ATP, or 0.16 0.16 [3H]ATP was bound by dnaB, dnaC(D), or the combination, as measured by Millipore binding, but more sensitive means 0.08 0.08 of measuring weak binding have not been used as yet. At- , -4.-i I tempts to determine if a small amount of ATP were hy- 4 8 12 16 20 24 28 32 drolyzed by the interaction of dnaB with dnaC(D) gene prod- Fraction Number uct were complicated by the ribonucleoside triphosphatase activity associated with dnaB (21), which, as shown below, agarose of FIG. 1. BioGel A-5m gel filtration dnaB, dnaC(D), dnaC(D) gene product inhibits. and the combination of gene products. (A) A reaction mixture The interaction of dnaB and dnaC(D) gene products de- (0.075 ml) containing dnaB (1.2 U), dnaC(D) (1.5 U), 0.5 mM pended on the ratio of these two proteins. In the experiment ATP, 1.0 mM MgCl2, 0.05 mg/ml of bovine serum albumin, 30 mM potassium phosphate (pH 6.0), and 10 mM dithiothreitol shown in Fig. 1A, half of the dnaC(D) activity recovered was was applied to a 0.5 X 20-cm column of BioGel A-5m agarose associated with dnaB.
Load more

Recommended publications
  • Replisome Assembly at Oric, the Replication Origin of E. Coli, Reveals an Explanation for Initiation Sites Outside an Origin
    Molecular Cell, Vol. 4, 541±553, October, 1999, Copyright 1999 by Cell Press Replisome Assembly at oriC, the Replication Origin of E. coli, Reveals an Explanation for Initiation Sites outside an Origin Linhua Fang,*§ Megan J. Davey,² and Mike O'Donnell²³ have not been addressed. For example, is the local un- *Microbiology Department winding sufficiently large for two helicases to assemble Joan and Sanford I. Weill Graduate School of Medical for bidirectional replication, or does one helicase need Sciences of Cornell University to enter first and expand the bubble via helicase action New York, New York 10021 to make room for the second helicase? The known rep- ² The Rockefeller University and licative helicases are hexameric and encircle ssDNA. Howard Hughes Medical Institute Which strand does the initial helicase(s) at the origin New York, New York 10021 encircle, and if there are two, how are they positioned relative to one another? Primases generally require at least transient interaction with helicase to function. Can Summary primase function with the helicase(s) directly after heli- case assembly at the origin, or must helicase-catalyzed This study outlines the events downstream of origin DNA unwinding occur prior to RNA primer synthesis? unwinding by DnaA, leading to assembly of two repli- Chromosomal replicases are comprised of a ring-shaped cation forks at the E. coli origin, oriC. We show that protein clamp that encircles DNA, a clamp-loading com- two hexamers of DnaB assemble onto the opposing plex that uses ATP to assemble the clamp around DNA, strands of the resulting bubble, expanding it further, and a DNA polymerase that binds the circular clamp, yet helicase action is not required.
    [Show full text]
  • Initiation of Enzymatic Replication at the Origin of the Escherichia
    Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3954-3958, June 1985 Biochemistry Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: Primase as the sole priming enzyme (DNA/orC/plasmids) ARIE VAN DER ENDEt, TANIA A. BAKER, TOHRU OGAWA*, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Arthur Kornberg, January 28, 1985 ABSTRACT The enzymatic replication of plasmids con- MATERIALS AND METHODS taining the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme DNAs and Reagents. pCM959 (4) was a gift from M. Meijer priming systems: primase alone, RNA polymerase alone, or (University of Amsterdam, The Netherlands); pTOA7 (T. both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Ogawa) was constructed by inserting the Hae II-Acc I Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, oriC-containing fragment from M13oriC26 (7) via EcoRI 3562-3566). At certain levels of auxiliary proteins linkers into EcoRI-cleaved pMAPCdSG10, a deletion deriva- (topoisomerase I, protein HU, and RNase H), the solo primase tive of pBR327 (W. A. Segraves, personal communication); system is efficient and responsible for priming synthesis of all pSY317, M13oriC26, M13oriC2LB5, and M13AE101 are DNA strands. Replication of oriC plasmids is here separated described in Table 1 and elsewhere (3, 7). Tricine, creatine into four stages: (i) formation of an isolable, prepriming phosphate, ribo- and deoxyribonucleoside triphosphates complex requiring oriC, dnaA protein, dnaB protein, dnaC (rNTPs and dNTPs) were from Sigma; a-32P-labeled dTTP, protein, gyrase, single-strand binding protein, and ATP; (ii) rATP, rUTP, rGTP, and rCTP (>400 Ci/mmol; 1 Ci = 37 formation of a primed template by primase; (iii) rapid, GBq) were from Amersham.
    [Show full text]
  • The Genetic Material: DNA the Central Dogma of Genetics
    The Genetic material: DNA The Central Dogma of Genetics DNA transcription Reverse tititranscriptio RNA n translation Protein • A, T, G, C in DNA • A, U, G, C in RNA • DNA is double stranded • DNA has po lar ity 5’ to 3’ • A T base pair, G C base pair • RNA is single stranded, also has polarity , generally referred as upstream and downstream. In RNA: A U base pair G C base pair. This type of base pa iri ng i n RNA causes secon dary st ruct ure. 1. 5’ vs 3’ 2. Purines vs. Pyrimidines 3. A vs. G 4. C vs. T 5. Transitions vs. transversions AT/GC ratios and their applications • Genome composition and chtitiharacterization • Implications in sequencing • Primer design • PCR yields • … The Polarity of DNA Higher order organization of genomes Most chromosomal DNA does not code for proteins or RNAs: e.g., Human genome 3 billion base pairs 25,000 genes x 2,000 bp per gene =5x10= 5 x 107 bp 5 x 107/3 x 109 = 1.67% MlMolecu lar P rob es: The tools of molecular genetics Concept of probes • “For diagnostic tests, the agent that is used to detect the presence of a molecule in the sample”. • “A DNA sequence that is used to detect the presence o f a comp lement ary sequence by hybridization with a nucleic acid sample”. Need for probes • Screen for the gene of interest • Southern blot to understand genomic structure and gene copy numbers • Northern blot for analysis of RNA expression • Verification of allelic amplification in PCR • ..
    [Show full text]
  • 1 Mechanistic Studies of DNA Replication, Lesion Bypass, And
    Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Austin Tyler Raper, B.S. The Ohio State University Biochemistry Graduate Program The Ohio State University 2018 Dissertation Committee Dr. Zucai Suo, Advisor Dr. Ross Dalbey Dr. Michael Poirier Dr. Richard Swenson 1 Copyrighted by Austin Tyler Raper 2018 2 Abstract DNA acts as a molecular blueprint for life. Adenosine, cytidine, guanosine, and thymidine nucleotides serve as the building blocks of DNA and can be arranged in near- endless combinations. These unique sequences of DNA may encode genes that when expressed produce RNA, proteins, and enzymes responsible for executing diverse tasks necessary for biological existence. Accordingly, careful maintenance of the molecular integrity of DNA is paramount for the growth, development, and functioning of organisms. However, DNA is damaged upon reaction with pervasive chemicals generated by normal cellular metabolism or encountered through the environment. The resulting DNA lesions act as roadblocks to high-fidelity A- and B-family DNA polymerases responsible for replicating DNA in preparation for cell division which may lead to programmed cell death. Additionally, these lesions may fool the polymerase into making errors during DNA replication, leading to genetic mutations and cancer. Fortunately, the cell has evolved DNA damage tolerance as an emergency response to such lesions. During DNA damage tolerance, a damage-stalled high-fidelity polymerase is substituted for a specialized Y-family polymerase, capable of bypassing the offending DNA lesion, for replication to continue.
    [Show full text]
  • A MUTYH Germline Mutation Is Associated with Small Intestinal
    248 J P Dumanski et al. MUTYH substitution 24:8 427–443 Research Gly396Asp in SI-NETs Open Access A MUTYH germline mutation is associated with small intestinal neuroendocrine tumors Jan P Dumanski1,*, Chiara Rasi1,*, Peyman Björklund2, Hanna Davies1, Abir S Ali3, Malin Grönberg3, Staffan Welin3, Halfdan Sorbye4,5, Henning Grønbæk6, Janet L Cunningham7, Lars A Forsberg1, Lars Lind8, Erik Ingelsson9, Peter Stålberg10, Per Hellman10 and Eva Tiensuu Janson3 1Department of Immunology, Genetics and Pathology and SciLifeLab, Uppsala University, Uppsala, Sweden 2Department of Surgical Sciences, Experimental Surgery, Uppsala University, Uppsala, Sweden 3Department of Medical Sciences, Endocrine Oncology, Uppsala University, Uppsala, Sweden 4Department of Oncology, Haukeland University Hospital, Bergen, Norway 5Department of Clinical Science, University of Bergen, Bergen, Norway 6Department of Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus, Denmark 7 Department of Neuroscience, Uppsala University, Uppsala, Sweden Correspondence 8 Department of Medical Sciences, Uppsala University, Uppsala, Sweden should be addressed 9 Division of Cardiovascular Medicine, Department of Medicine, Stanford University, San Francisco, California, USA to E Tiensuu Janson 10 Department of Surgical Sciences, Endocrine Surgery, Uppsala University, Uppsala, Sweden Email *(J P Dumanski and C Rasi shared first co-authorship) eva.tiensuu_janson@medsci. uu.se Abstract The genetics behind predisposition to small intestinal neuroendocrine tumors (SI-NETs) Key Words Endocrine-Related Cancer Endocrine-Related is largely unknown, but there is growing awareness of a familial form of the disease. f familial cancer We aimed to identify germline mutations involved in the carcinogenesis of SI-NETs. f cancer predisposition The strategy included next-generation sequencing of exome- and/or whole-genome of f small intestinal carcinoid blood DNA, and in selected cases, tumor DNA, from 24 patients from 15 families with f oxidative stress the history of SI-NETs.
    [Show full text]
  • Distinct Co-Evolution Patterns of Genes Associated to DNA Polymerase III Dnae and Polc Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3*
    Engelen et al. BMC Genomics 2012, 13:69 http://www.biomedcentral.com/1471-2164/13/69 RESEARCHARTICLE Open Access Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC Stefan Engelen1,2, David Vallenet2, Claudine Médigue2 and Antoine Danchin1,3* Abstract Background: Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results: Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co- evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion: DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.
    [Show full text]
  • Glycolytic Pyruvate Kinase Moonlighting Activities in DNA Replication
    Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere To cite this version: Steff Horemans, Matthaios Pitoulias, Alexandria Holland, Panos Soultanas, Laurent Janniere. Gly- colytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation. 2020. hal-02992157 HAL Id: hal-02992157 https://hal.archives-ouvertes.fr/hal-02992157 Preprint submitted on 10 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Glycolytic pyruvate kinase moonlighting activities in DNA replication initiation and elongation Steff Horemans1, Matthaios Pitoulias2, Alexandria Holland2, Panos Soultanas2¶ and Laurent Janniere1¶ 1 : Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France 2 : Biodiscovery Institute, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK Short title: PykA moonlighting activity in DNA replication Key Words: DNA replication; replication control; central carbon metabolism; glycolytic enzymes; replication enzymes; cell cycle; allosteric regulation. ¶ : Corresponding authors Laurent Janniere: [email protected] Panos Soultanas : [email protected] 1 SUMMARY Cells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions.
    [Show full text]
  • The Molecular Coupling Between Substrate Recognition and ATP Turnover in A
    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader Neha Puri1,2, Amy J. Fernandez1, Valerie L. O’Shea Murray1,3, Sarah McMillan4, James L. Keck4, James M. Berger1,* 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205 2Bristol Myers Squibb, 38 Jackson Road, Devens, MA 01434 3Saul Ewing Arnstein & Lehr, LLP, Centre Square West, 1500 Market Street, 38th Floor, Philadelphia, PA 19102 4Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706 *Corresponding author Email: [email protected] Keywords: DNA replication, AAA+ ATPase, Helicase, Meier-Gorlin Syndrome 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood.
    [Show full text]
  • Conversion of OX174 and Fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia Coli (Dnac, Dnad, and Dnag Gene Products/DNA Polymerase III) REED B
    Proc. Nat. Acad. Sci. USA Vol. 69, No. 11, pp. 3233-3237, November 1972 Conversion of OX174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli (dnaC, dnaD, and dnaG gene products/DNA polymerase III) REED B. WICKNER, MICHEL WRIGHT, SUE WICKNER, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by Harry Eagle, August 28, 1972 ABSTRACT 4X174 and M13 (fd) single-stranded cir- MATERIALS AND METHODS cular DNAs are converted to their replicative forms by ex- tracts of E. coli pol Al cells. We find that the qX174 DNA- [a-32P]dTTP was obtained from New England Nuclear Corp. dependent reaction requires Mg++, ATP, and all four de- OX174 DNA was prepared by the method of Sinsheimer (7) oxynucleoside triphosphates, but not CTP, UTP, or GTP. or Franke and Ray (8), while fd viral DNA was prepared as This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, described (9). Pancreatic RNase was the highest grade ob- but not that of dnaF (ribonucleotide reductase). The in tainable from Worthington Biochemical Corp. It was further vitro conversion of fd single-stranded DNA to the replica- freed of possible contaminating DNase by heating a solution tive form requires all four ribonucleoside triphosphates, (2 mg/ml in 15 mM sodium citrate, pH 5) at 800 for 10 min. Mg++, and all four deoxynucleoside triphosphates. The E. protein was purified from E. coli strain reaction involves the product of gene dnaE but not those coli unwinding of genes dnaC, dnaD, dnaF, or dnaG.
    [Show full text]
  • Consequences and Resolution of Transcription–Replication Conflicts
    life Review Consequences and Resolution of Transcription–Replication Conflicts Maxime Lalonde †, Manuel Trauner †, Marcel Werner † and Stephan Hamperl * Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, 81377 Munich, Germany; [email protected] (M.L.); [email protected] (M.T.); [email protected] (M.W.) * Correspondence: [email protected] † These authors contributed equally. Abstract: Transcription–replication conflicts occur when the two critical cellular machineries respon- sible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these en- counters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication. Citation: Lalonde, M.; Trauner, M.; Keywords: transcription–replication conflicts; genomic instability; R-loops; torsional stress; common Werner, M.; Hamperl, S. fragile sites; early replicating fragile sites; replication stress; chromatin; fork reversal; MIDAS; G-MiDS Consequences and Resolution of Transcription–Replication Conflicts. Life 2021, 11, 637. https://doi.org/ 10.3390/life11070637 1.
    [Show full text]
  • The Functional Analysis of Yaba, Which Interacts with Dnaa and Regulates Initiation of Chromosome Replication in Bacillus Subtils
    Genes Genet. Syst. (2008) 83, p. 111–125 The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils Eunha Cho, Naotake Ogasawara and Shu Ishikawa* Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan (Received 15 January 2008, accepted 1 February 2008) The initiation of bacterial chromosome DNA replication and its regulation are critical events. DnaA is essential for initiation of DNA replication and is con- served throughout bacteria. In Escherichia coli, hydrolysis of ATP-DnaA is pro- moted by Hda through formation of a ternary complex with DnaA and DnaN, ensuring the timely inactivation of DnaA during the replication cycle. In Bacillus subtilis, YabA also forms a ternary complex with DnaA and DnaN, and negatively regulates the initiation step of DNA replication. However, YabA shares no struc- tural homology with Hda and the regulatory mechanism itself has not been clarified. Here, in contrast to Hda, we observed that dnaA transcription was stable during under- and overexpression of YabA. ChAP-chip assays showed that the depletion of YabA did not affect DNA binding by DnaA. On the other hand, yeast two-hybrid analysis indicated that the DnaA ATP-binding domain interacts with YabA. Moreover, mutations in YabA interaction-deficient mutants, isolated by yeast two-hybrid analysis, are located at the back of the ATP-binding domain, whereas Hda is thought to interact with the ATP-binding pocket itself. The intro- duction into B. subtilis of a dnaAY144C mutation, which disabled the interaction with YabA but did not affect interactions either with DnaA itself or with DnaD, resulted in over-initiation and asynchronous initiation of replication and disabled the formation of YabA foci, further demonstrating that the amino acid on the oppo- site side to the ATP-binding pocket is important for YabA binding.
    [Show full text]
  • Ordered Association of Helicase Loader Proteins with the Bacillus Subtilis Origin of Replication in Vivo
    Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Smits, Wiep Klaas, Alexi I. Goranov, and Alan D. Grossman. “Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo.” Molecular Microbiology 75.2 (2010): 452–461. As Published http://dx.doi.org/10.1111/j.1365-2958.2009.06999.x Publisher Wiley Blackwell (Blackwell Publishing) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/73616 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike 3.0 Detailed Terms http://creativecommons.org/licenses/by-nc-sa/3.0/ NIH Public Access Author Manuscript Mol Microbiol. Author manuscript; available in PMC 2011 January 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Mol Microbiol. 2010 January ; 75(2): 452±461. doi:10.1111/j.1365-2958.2009.06999.x. Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo Wiep Klaas Smits, Alexi I. Goranov, and Alan D. Grossman* Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Summary The essential proteins DnaB, DnaD, and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order.
    [Show full text]