DOCSLIB.ORG

Genetic Evidence for Two Protein Domains and a Potential New Activityin Bacteriophage T4 DNA Polymerase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Genetic Evidence for Two Protein Domains and a Potential New Activityin Bacteriophage T4 DNA Polymerase Copyright 0 1990 by the Genetics Society of America Genetic Evidence for Two Protein Domains and a Potential New Activityin Bacteriophage T4 DNA Polymerase Linda J. Reha-Krantz Department of Genetics, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Manuscript received June 22, 1989 Accepted for publication October 7, 1989 ABSTRACT Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal,temperature-sensitive (ts) mu- tator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing func- tions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5’ + 3’ exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, .. ” contains a discrete N-terminal domain. ANY DNA polymerases from diverse organisms parisons between E. coli DNA pol I and bacteriophage M (e.g., human; yeast; viruses-herpes, adeno, vac- T4 DNA polymerase, and, by inference, toother cinia; and bacteriophage-T4, 429, PRD1) share sev- DNA polymerases. Many DNA polymerases contain eral regions of colinear protein sequence homology N-terminal protein sequences upstream from the pro- (SPICERet al. 1988; BERNARDet al. 1987; JUNG et al. posed 3’ += 5’ exonuclease and polymerase domains. 1987; WANG,WONG and KORN 1989). DNA polym- In E. coli DNA pol I, the N terminus encodes a discrete erase I (pol I) from Escherichia coli may contain just protein domain with 5’ + 3‘ exonuclease activity that one of the conserved sequences, a part of the most N- can be separated by mild proteolysis from the larger terminal conserved region (REHA-KRANTZ 1988a,b; Klenow fragment (3’ + 5’ exonuclease and polym- 1989; SPICERet al. 1988). Although the protein se- erase domains) (LEHMAN andCHIEN 1973; JACOBSEN, quence similarity involves only a few amino acids, KLENOW andOVERGAARD-HANSEN 1974). Some N- these amino acids in E. coli DNA pol I form part of terminal T4 DNA polymerase protein sequences are the 3’ + 5’ exonuclease active site (JOYCE and STEITZ similar to sequences in the E. coli DNA polymerase I 1987;DERBYSHIRE et al. 1988). Thus, the most N- 5’ + 3’ exonuclease domain; there is also some pro- terminal conserved region in the related DNA polym- tein sequence similarity with E. coli RNase H. Fur- erases, called region IV by WANG,WONG and KORN thermore, intragenic complementation has been de- tected between T4 N-terminal DNA polymerase mu- (1 989)is likely the location of active or cryptic 3’ + tantsand DNA polymerase C-terminal mutants. 5’ exonuclease function(REHA-KRANTZ 1988a, b; Intragenic complementation has also been observed 1989). The C-terminal conserved sequences are pre- with some herpes (HSV-1) N-terminal DNA polym- dicted to encode polymerizing functions such as de- erase mutants (CHARTRANDet al. 1980; WELLERet al. oxynucleoside triphosphate bindingand DNA binding 1983; A. I. MARCYand D.M. COEN,personal com- (GIBBSet al. 1985, 1988; SPICERet al. 1988; WANG, munication). The positive complementation results WONG and KORN 1989;REHA-KRANTZ 1988a, b). suggest that T4 and HSV-1 DNA polymerases may Polymerizing functions are also located in the C ter- have discrete, N-terminal domains while the protein minus of E. coli DNA pol I (JOYCE and STEITZ1987). sequence similarities suggest that the N terminus may Thus, although there is little proteinsequence ho- encode a 5’ + 3’ exonuclease or RNase H-type activ- mology shared between E. coli DNA pol I and the ity; an RNase H-type activity has recently been de- above DNA polymerases, many DNA polymerases tected in the HSV-1 DNA polymerase (CRUTEand appear to have the same relative organization of 3’ + LEHMAN 1989;MARCY et al. 1989). The main predic- 5’ exonuclease and polymerase activities. (REHA- tion arising fromthese studies is that many DNA KRANTZ1988a, b; BERNADet al. 1989; LEAVITTand polymerases may share the same structural-functional IT0 1989). organization as found in E. coli DNA polymerase I: a Arguments are presented here that extend com-the distinct N-terminal domain that may encode a 5’ + 3’ exonuclease or RNase H activity adjacentto a The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “advertisement” Klenow-type domain with 3’ + 5’ exonuclease and in accordance with 18 U.S.C. $1734 solely to indicate this fact. polymerase activities. Genetlcs 124: 213-220 (February, 1990) 214 L. J. Reha-Krantz MATERIALS AND METHODS TABLE 1 Phage and bacterial strains: Phage T4 and bacterial Complementation betweenT4 DNA polymerase (g43) alleles strainshave been described (REHA-KRANTZet al. 1986; - ~~ ~~ REHA-KRANTZ 1988a). Phage Mutation and site" Burst sile' Complementation tests: Single and mixed infections at a Single phage infections multiplicity of five foreach phage strain were doneat Wild type 64.0 nonpermissiveconditions: high tem erature(43") which amE4314 Trpamber 202 0.06 restricts ts phage andhost E. coli B (suB ) which does not have amE4306 Trpamber 20.004 13 a nonsense suppressortRNA andrestricts phage with amber amB22 0.005Glnamber 731 mutations. Many gene 43 amber mutants aresuppressed at amE4302 amber844 Trp 0.01 low levels on suo hosts at 30" by translationalambiguity tsmeA62 Gly 82 Asp 0.03 (KARAM and O'DONNELL 1973).At 43", however, there is tsL56 Ala 89 Thr, Asp 363 Asn 0.04 little suppression (see discussion on transmission coefficients tsPS7 Asp 112 Asn 0.01 below). At 10 min post infection, cultures were diluted 100- tSPN Y 123 Pro Leu 0.09 fold into 43" broth to diluteaway from unadsorbed phage. tsmel5 Asp 131 Cly 0.08 Chloroform was added at 40 min and the progeny yields tsA6O 0.07 SerGly 172 were determined by plating on CR63 (su+) at 30". Progeny tsA 73 225 Arg His 0.02 are expectedonly if the infecting phage can mutually supply tsMl9 ProLeu 340 0.01 in trans the function missing in the othercoinfecting phage. tsSY Met 671 Ile 0.03 The burst size is equal to the total number of progeny Mixed infections: divided by the total number of infected bacteria. The infec- tsmel62 + amB22 0.06 tive center titer of thewild-type control was used to calculate tsL56 + amB22 0.05 all burst sizes. Parental phage were recovered in all cases of tsPS7 + amB22 0.5 positive complementation. tsmel5 + tsL56 0.6 Transmission coefficients: In order to determine the tsmel5 + tsme162 0.5 level of suppression due to translational ambiguity at 43" in tsPS7 + tsmel62 0.3 E. coli B, dilutions of the amber phage-E. coli B infective tsmel5 + tsPS99 0.8 centers were mixed at 10 min postinfection with the su+ tsA60 + amE4314 0.2 host CR63 in soft agar, then overlaid on hard agar and tsA60 + amB22 0.7 incubated overnight at 43". Any viable phage produced in tsA6O + amE4302 0.1 the amber phage-E. coli B infective centers infect the sur- tsA73 + amE4314 1.4 rounding CR63 bacteria and produce plaques. No plaques tsA73 + amE4302 0.1 were observed from amE4314, amE4306 and amE4302 in- tsmel5 + tsA60 70.0 fective centers, but 10% of the amB22-E. coli B infective tsPS99 + tsA60 11.0 centers produced plaques. Thus, low levels of suppression tsmel5 + tsA73 31.0 cannotaccount for the positive complementation results tsPS99 + tsA73 8.0 observed with amE4314, amE4306 and amE4302 infections. tsPS99 + tsMlY 5.0 Suppression is also probably not a factor in the positive tsPSY9 + tsS9 30.0 complementation results observed with amB22. For comple- tsPS99 + amE4314 28.0 mentation tests, only 40 min was allowed to produce viable tsPS99 + amE4306 19.0 phage and few progeny were detected in amB22 infections tsPS9Y + amB22 11.0 (Table 1). Thus, apparently a growth cycle longer then 40 tsPS99 + amE3402 10.0 min is required to produce progeny from amB22-infected tsmel5 + amE4314 13.5 E. coli B cells. tsme15 + amE4306 11.0 Alkaline sucrose gradients: Pulse-labeled DNA was pre- tsmel5 + amB22 11.0 pared as described (KONRADand LEHMAN 1974). E. coli tsmel5 + amE4302 8.0 K12SH28 (FANGMAN 1969),which has reduced thymidine Extragenic complementation: phosphorylase acitivity, was grown at 43" to 2 X lo8 bacte- amB22 + amN122 (gene 42) 46.0 ria/ml in M9 mediumsupplemented with 0.4% glucose, 0.5% casamino acids, and 0.01% MgS04. At to, cells were Standard three letter amino acid code is used. The wild-type amino acid is given first followed by the codon position and then infected with phage at a multiplicity of ten. At t5 min, the the mutant aminoacid. Mutation identifications are given in REHA- infected cells were diluted tenfold into 10ml of prewarmed KRANTZ(1 988a) and REHA-KRANTZ(1 989). M9 medium. At t7 min, 200 p1 of ["]thymidine (0.3 mCi/ *Burst size is equal tothe total number of progenyphage ml were added. Cultures were aerated by shaking through- produced in one cycle of replication divided by the total number of out infection and labeling procedures. Incorporation was infected bacteria. All infections were at 43" in host E. coli B (see quenched at 10 sec, 45 sec, 2 min and 10 min by adding an MATERIALS AND METHODS). equalvolume of 75%ethanol solution containing 2% phenol, 20 mM sodium acetate (pH 5.2), and 2 mM EDTA. pUCl8 plasmid DNA; the peak of activity was recovered in Cells were centrifuged at 15,000 X g for 10 min and the fraction 22. Gradient samples, 200 pl, were collected from pellet was resuspended in 0.3 ml of 0.2 M NaOH-10 mM the bottom and precipitated with 10% trichloroacetic acid. EDTA and incubated at 40" for 1 hr.
Load more

Recommended publications
  • Mutations That Separate the Functions of the Proofreading Subunit of the Escherichia Coli Replicase
    G3: Genes|Genomes|Genetics Early Online, published on April 15, 2015 as doi:10.1534/g3.115.017285 Mutations that separate the functions of the proofreading subunit of the Escherichia coli replicase Zakiya Whatley*,1 and Kenneth N Kreuzer*§ *University Program in Genetics & Genomics, Duke University, Durham, NC 27705 §Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 1 © The Author(s) 2013. Published by the Genetics Society of America. Running title: E. coli dnaQ separation of function mutants Keywords: DNA polymerase, epsilon subunit, linker‐scanning mutagenesis, mutation rate, SOS response Corresponding author: Kenneth N Kreuzer, Department of Biochemistry, Box 3711, Nanaline Duke Building, Research Drive, Duke University Medical Center, Durham, NC 27710 Phone: 919 684 6466 FAX: 919 684 6525 Email: [email protected] 1 Present address: Department of Biology, 300 N Washington Street, McCreary Hall, Campus Box 392, Gettysburg College, Gettysburg, PA 17325 Phone: 717 337 6160 Fax: 7171 337 6157 Email: [email protected] 2 ABSTRACT The dnaQ gene of Escherichia coli encodes the ε subunit of DNA polymerase III, which provides the 3’ 5’ exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of ε leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our lab identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response following quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby in addition to proofreading, ε plays a distinct role in replisome disassembly and/or processing of stalled replication forks.
    [Show full text]
  • DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine
    Annu. Rev. Biochem. 1995.64:171-200 Copyright Ii) 1995 byAnnual Reviews Inc. All rights reserved DNA POLYMERASE III HOLOENZYME: Structure and Function of a Chromosomal Replicating Machine Zvi Kelman and Mike O'Donnell} Microbiology Department and Hearst Research Foundation. Cornell University Medical College. 1300York Avenue. New York. NY }0021 KEY WORDS: DNA replication. multis ubuni t complexes. protein-DNA interaction. DNA-de penden t ATPase . DNA sliding clamps CONTENTS INTRODUCTION........................................................ 172 THE HOLO EN ZYM E PARTICL E. .......................................... 173 THE CORE POLYMERASE ............................................... 175 THE � DNA SLIDING CLAM P............... ... ......... .................. 176 THE yC OMPLEX MATCHMAKER......................................... 179 Role of ATP . .... .............. ...... ......... ..... ............ ... 179 Interaction of y Complex with SSB Protein .................. ............... 181 Meclwnism of the yComplex Clamp Loader ................................ 181 Access provided by Rockefeller University on 08/07/15. For personal use only. THE 't SUBUNIT . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 182 Annu. Rev. Biochem. 1995.64:171-200. Downloaded from www.annualreviews.org AS YMMETRIC STRUC TURE OF HOLO EN ZYM E . 182 DNA PO LYM ER AS E III HOLO ENZ YME AS A REPLIC ATING MACHINE ....... 186 Exclwnge of � from yComplex to Core .................................... 186 Cycling of Holoenzyme on the LaggingStrand
    [Show full text]
  • Tetrameric Uvrd Helicase Is Located at the E. Coli Replisome Due to Frequent Replication Blocks Adam J
    bioRxiv preprint doi: https://doi.org/10.1101/2021.02.22.432310; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks Adam J. M Wollman1,2,3,, Aisha H. Syeda1,2, Andrew Leech4 , Colin Guy5, 6, Peter McGlynn2,6, Michelle Hawkins2 and Mark C. Leake1,2 The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 1 Department of Physics, University of York, York YO10 5DD, United Kingdom. 2 Department of Biology, University of York, York YO10 5DD, United Kingdom. 3 Current address: Biosciences Institute, Newcastle University, NE1 7RU, United Kingdom. 4 Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, United Kingdom 5 Current address: Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, United Kingdom 6 Previous address: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom * To whom correspondence should be addressed. To whom correspondence should be addressed. Tel: +44 (0)1904322697. Email: [email protected] Present Address: Departments of Physics and Biology, University of York, York YO10 5DD, United Kingdom ABSTRACT DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help replication machinery overcome blocks by removing incoming nucleoprotein complexes or aiding the re-initiation of replication.
    [Show full text]
  • Exonucleolytic Editing by DNA Polymerase III Holoenzyme- (DNA Replication/Fidelity of Replication/Mutagenesis/Proofreading) HARRISON Echolstt, CHI Lutf, and PETER M
    Proc. NatL Acad. Sci. USA Vol. 80, pp. 2189-2192, April 1983 Biochemistry Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme- (DNA replication/fidelity of replication/mutagenesis/proofreading) HARRISON ECHOLStt, CHI Lutf, AND PETER M. J. BURGERSt§ tDeartament of Molecular Biology, University of California, Berkeley, California 94720; and tDepartment of Biochemistry, Stanford University School of Medicine, StOrd. California 94305 Communicated by I. Robert Lehman, January 17, 1983 ABSTRACT The closely linked mutD and dnaQ mutations clease. We infer that the mutD (dnaQ) gene product controls confer a vastly increased mutation rate on Escherichia coli and the editing capacity of pol III. thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene MATERIALS AND METHODS product, we have measured the 3' -* 5' exonucleolytic editing ac- tivity of polymerase m holoenzyme from mutD5 and dnaQ49 mu- Materials. Unlabeled deoxynucleoside triphosphates and the tants. The editing activities of the mutant enzymes are defective polymers (dA)1,5oo and (dT)17 were obtained from P-L Bio- compared to wild type, as judged by two assays: (i) decreased ex- chemicals. [3H]dTTP and [3H]dTMP were purchased from New cision of a terminal mispaired base from a copolymer substrate England Nuclear and Schwarz/Mann, respectively. [a-32P]dTTP and (i) turnover of dTTP to dTMP during replication with a phage was obtained from Amersham. Polyethylenimine (PEI)-cellu- G4 DNA template. Thus, the mutD (dnaQ). gene product is likely lose plates were from Machery-Nagel and DEAE-paper (DE81) to control the editing (proofreading) capacity of polymerase HI was from Whatman.
    [Show full text]
  • Q 297 Suppl USE
    The following supplement accompanies the article Atlantic salmon raised with diets low in long-chain polyunsaturated n-3 fatty acids in freshwater have a Mycoplasma dominated gut microbiota at sea Yang Jin, Inga Leena Angell, Simen Rød Sandve, Lars Gustav Snipen, Yngvar Olsen, Knut Rudi* *Corresponding author: [email protected] Aquaculture Environment Interactions 11: 31–39 (2019) Table S1. Composition of high- and low LC-PUFA diets. Stage Fresh water Sea water Feed type High LC-PUFA Low LC-PUFA Fish oil Initial fish weight (g) 0.2 0.4 1 5 15 30 50 0.2 0.4 1 5 15 30 50 80 200 Feed size (mm) 0.6 0.9 1.3 1.7 2.2 2.8 3.5 0.6 0.9 1.3 1.7 2.2 2.8 3.5 3.5 4.9 North Atlantic fishmeal (%) 41 40 40 40 40 30 30 41 40 40 40 40 30 30 35 25 Plant meals (%) 46 45 45 42 40 49 48 46 45 45 42 40 49 48 39 46 Additives (%) 3.3 3.2 3.2 3.5 3.3 3.4 3.9 3.3 3.2 3.2 3.5 3.3 3.4 3.9 2.6 3.3 North Atlantic fish oil (%) 9.9 12 12 15 16 17 18 0 0 0 0 0 1.2 1.2 23 26 Linseed oil (%) 0 0 0 0 0 0 0 6.8 8.1 8.1 9.7 11 10 11 0 0 Palm oil (%) 0 0 0 0 0 0 0 3.2 3.8 3.8 5.4 5.9 5.8 5.9 0 0 Protein (%) 56 55 55 51 49 47 47 56 55 55 51 49 47 47 44 41 Fat (%) 16 18 18 21 22 22 22 16 18 18 21 22 22 22 28 31 EPA+DHA (% diet) 2.2 2.4 2.4 2.9 3.1 3.1 3.1 0.7 0.7 0.7 0.7 0.7 0.7 0.7 4 4.2 Table S2.
    [Show full text]
  • Escherichia Coli Dnax Product, the 7 Subunit of DNA Polymerase
    Proc. Natl. Acad. Sci. USA Vol. 84, pp. 2713-2717, May 1987 Biochemistry Escherichia coli DnaX product, the 7 subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity (DNA replication/dnaZX gene) SUK-HEE LEE AND JAMES R. WALKER Department of Microbiology, University of Texas, Austin, TX 78712 Communicated by Esmond E. Snell, January 7, 1987 ABSTRACT The dnaZX gene of Escherichia coli directs Germino et al. (14) have used affinity chromatography to the synthesis of two proteins, DnaZ and DnaX. These products purify a bifunctional fusion protein consisting of the initiator are confirmed as the y and X subunits of DNA polymerase HI of plasmid R6K replication fused near its C-terminal end to because antibody to a synthetic peptide present in both the ,B-galactosidase. DnaZ and DnaX proteins reacts also with the y and T subunits ofholoenzyme. To characterize biochemically the Tsubunit, for which there has been no activity assay, the dnaZX gene was METHODS fused to the 13-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (T) Bacterial Strains and Plasmids. Strain RB791, a lacIQ L8 were replaced by fi-galactosidase lacking only 7 N-terminal derivative of strain W3110 (15), was obtained from Nina amino acids. The 185-kDa fusion protein, which retained Irwin (Harvard University). Strain M182, A(lacIPOZYA)- ,8-galactosidase activity, was overproduced to the level ofabout X74, galK, galU, rpsL (16), was obtained from Richard 5% of the soluble cellular protein by placing the gene fusion Meyer (University of Texas).
    [Show full text]
  • Using Genomics to Study Susceptibility and Resistance To
    Integrating Undergraduates into Educationally Meaningful Research Programs and Courses Jeffrey H. Miller University of California, Los Angeles CA Challenge Involve Students in Research Goals 1. Transmit the excitement of tackling the unknown 2. Have rapid rewards instead of long apprenticeship 3. Present an intellectual process 4. Student has ownership of a project and results, not a “cog in the wheel” 5. Understand the practical importance of the relevant area of research Requirements • Commitment from mentor • Commitment from student • Projects that allow goals to be achieved Environmental Samples Collected and Source: Strain Species Color Source CB001 E. coli strain Pinkish red Lab contaminant CB002 Staphylococcus strain White Amie Fong finger CB003 Bacillus cereus Salmon Soil CB004 Bacillus cereus Pale yellow Soil CB005 Bacillus cereus Yellow orange Soil CB006 Bacillus cereus Orange Soil CB007 Bacillus cereus Yellow Amie Fong finger CB008 Bacillus cereus Neon red Amie Fong hair CB009 E. fergusonii (potentially) Bloody red/orange South campus dumpster CB010 Bacillus cereus Baby pink UCLA campus CB011 Staphylococcus hominis Tan Arrowhead water jug from CHS CB012 Bacillus cereus Bright red Orthopaedic hospital bathroom handle CB013 Staphylococcus strain White #2 Orthopaedic hospital bathroom handle 8 12 11 1 13 7 10 2 M. Luteus 6 3 recC/tolC 9 5 4 Bacterial Art - Sydney Brenner WT 1. tolC 2. tolC recC Strain A=WT (yellow) starting mixture 1:20 Strain A: Strain B Strain B=Drug sensitive purple mutant (purple) growth without drug growth
    [Show full text]
  • A RAP 1-Interacting Protein Involved in Transcriptional Silencing and Telomere Length Regulation
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press A RAP 1-interacting protein involved in transcriptional silencing and telomere length regulation Christopher F.J. Hardy/ Lori Sussel, and David Shore^ Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032 USA The yeast RAPl protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAPl is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(Ci_3A) sequences. Previous studies have indicated that the regulatory function of RAPl is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAPl-interacting factor (RIFl). Strains carrying gene disruptions of RIFl grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rapl" mutants. Furthermore, hybrid proteins containing rapl^ missense mutations are defective in an interaction with RIFl in the two-hybrid system. Taken together, these data support the idea that the rapT phenotypes are attributable to a failure to recruit RIFl to silencers and telomeres and suggest that RIFl is a cofactor or mediator for RAPl in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIFl that partially restores the interaction with rapl** mutant proteins.
    [Show full text]
  • Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Research Online University of Wollongong Research Online Faculty of Science - Papers (Archive) Faculty of Science, Medicine and Health 2012 Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Andrew Robinson University of Wollongong, [email protected] Rebecca J. Causer University of Wollongong, [email protected] Nicholas E. Dixon University of Wollongong, [email protected] Follow this and additional works at: https://ro.uow.edu.au/scipapers Part of the Life Sciences Commons, Physical Sciences and Mathematics Commons, and the Social and Behavioral Sciences Commons Recommended Citation Robinson, Andrew; Causer, Rebecca J.; and Dixon, Nicholas E.: Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target 2012, 352-372. https://ro.uow.edu.au/scipapers/2996 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target Abstract "New antibiotics with novel modes of action are required to combat the growing threat posed by multi- drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad- spectrum antibacterials.
    [Show full text]
  • Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target
    352 Current Drug Targets, 2012, 13, 352-372 Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target Andrew Robinson, Rebecca J. Causer and Nicholas E. Dixon* School of Chemistry, University of Wollongong, Australia Abstract: New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets.
    [Show full text]
  • Fate of the Replisome Following Arrest by UV-Induced DNA Damage in Escherichia Coli
    Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli H. Arthur Jeiranian, Brandy J. Schalow, Charmain T. Courcelle, and Justin Courcelle1 Department of Biology, Portland State University, Portland, OR 97201 Edited by Mike E. O’Donnell, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, and approved May 24, 2013 (received for review January 18, 2013) Accurate replication in the presence of DNA damage is essential to allowing the machinery to overcome specific challenges such as genome stability and viability in all cells. In Escherichia coli, DNA collisions with the transcription apparatus or DNA-bound pro- replication forks blocked by UV-induced damage undergo a partial teins (1, 12, 13). In this study, we used thermosensitive replication resection and RecF-catalyzed regression before synthesis resumes. mutants to characterize how the composition of the replisome These processing events generate distinct structural intermediates changes following encounters with UV-induced photoproducts, a on the DNA that can be visualized in vivo using 2D agarose gels. biologically relevant lesion that is known to block the progression However, the fate and behavior of the stalled replisome remains of the replisome when located in the leading strand template (6, a central uncharacterized question. Here, we use thermosensitive 14–16). The results demonstrate that the DNA polymerases can mutants to show that the replisome’s polymerases uncouple and dissociate from the replisome in a modular manner without transiently dissociate from the DNA in vivo. Inactivation of α, β,or compromising the integrity of the replication fork. Dissociation of τ subunits within the replisome is sufficient to signal and induce the DNA polymerase from the replisome is sufficient and can the RecF-mediated processing events observed following UV dam- serve to initiate the processing of the replication fork DNA via the age.
    [Show full text]
  • III of Escherichia Coli: Characterization of a Hole Mutant and Comparison with a Dnaq (6-Subunit) Mutant STEVEN C
    JOURNAL OF BACTERIOLOGY, Feb. 1994, p. 815-821 Vol. 176, No. 3 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology holE, the Gene Coding for the 0 Subunit of DNA Polymerase III of Escherichia coli: Characterization of a holE Mutant and Comparison with a dnaQ (6-Subunit) Mutant STEVEN C. SLATER,'t MIRIAM R. LIFSICS,1 MIKE O'DONNELL,2'3 AND RUSSELL MAURER'* Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960,1 and Department of Microbiology, Cornell University Medical College,2 and Howard Hughes Medical Institute,3 New York, New York 10021 Downloaded from Received 4 October 1993/Accepted 2 December 1993 DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an arO heterotrimer that incorporates both a polymerase subunit ((; dnaE) and a proofreading subunit (£; dnaQ). The role of 0 is unknown. Here, we describe a null mutation ofholE, the gene for 0. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting a which was identical to a http://jb.asm.org/ suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the 0 subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of e mutants cannot be explained on the basis of interference with 0 function.
    [Show full text]