DOCSLIB.ORG

Fate of the Replisome Following Arrest by UV-Induced DNA Damage in Escherichia Coli

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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. By contrast, the helicase–primase complex (DnaB and DnaG) RecF pathway, similar to that seen when replication is arrested remains critically associated with the fork, leading to a loss of fork by UV-induced damage. By comparison, the helicase complex integrity, degradation, and aberrant intermediates when disrupted. remains associated with the replication fork throughout the re- The results reveal a dynamic replisome, capable of partial disassem- covery process. If the helicase is disrupted, aberrant intermediates, bly to allow access to the obstruction, while retaining subunits that degradation, and loss of fork integrity ensue. We propose that the CELL BIOLOGY maintain fork licensing and direct reassembly to the appropriate retention of the helicase is needed to maintain licensing for the location after processing has occurred. replication fork and direct reassembly to the appropriate location after processing has occurred. replication fork processing | RecF pathway Results he replisome consists of several, multisubunit protein com- Dissociation of the Polymerase from the Replisome Is Sufficient to Tplexes and is responsible for duplicating the genome. In Induce Processing Events Similar to Those Observed After UV-Induced Escherichia coli, it is comprised of three DNA polymerase com- Arrest. A schematic of each of the components of the replisome A plexes tethered to the DNA template by dimeric processivity tested in this study and their function is presented in Fig. 1 . factors, a τ complex that couples leading and lagging strand Temperature-sensitive mutants exist in subunits from each of – replisome’s complexes for which viability or functionality is sup- synthesis, and a helicase primase complex that separates the B duplex DNA and primes lagging strand synthesis (1–3). ported at 30 °C, but not at 42 °C (Fig. 1 ). Although replica- When the replisome encounters DNA damage that blocks its tion proceeds normally at the permissive temperature, it rapidly progression, the potential for mutagenesis, rearrangements, and decreases following inactivation of the thermosensitive protein at fi the restrictive temperature, similar to that seen after UV irradi- lethality increases signi cantly. Replication in the presence of C DNA damage can generate mutations if the wrong base is in- ation (Fig. 1 ). The exception to this is in the proofreading sub- unit e, encoded by dnaQts, which is mutagenic at the restrictive corporated, rearrangements if it resumes from the wrong site, temperature, but is not essential for viability or replication (17). or lethality if the obstructing lesion cannot be overcome. Follow- To determine how the replisome behaves or is modified fol- ing the arrest of replication at UV-induced damage, the nascent lowing encounters with DNA damage in vivo, these thermo- lagging strand is partially resected by the combined action of the sensitive mutants were used to deliberately disrupt the specific RecQ helicase and RecJ nuclease (4, 5). RecF-O-R, along with components of the replisome. We then compared the effect that RecA, limit this degradation and promote a transient regression the loss of that component had on replication processing to that of the DNA branch point, which is thought to be important for seen when replication encounters DNA damage. Replication and restoring the damaged region to a form that can be acted on by – processing intermediates were visualized in vivo on replicating repair enzymes or translesion DNA polymerases (4 10). These fragments of the plasmid pBR322 (Fig. 2). This plasmid replicates processing events generate distinct structural intermediates on using the host’s replication machinery, it can be linearized at its the DNA that can be readily visualized using 2D agarose gel single origin of replication, and it maintains a moderate copy analysis, a technique that allows one to identify the shape and number, making it useful to detect rare events such as replication structure of DNA molecules (5, 11). through a defined fragment (18). In this type of analysis, actively Although the processing that occurs on the DNA is well char- acterized, little is known about the behavior or composition of the replisome itself during these events. If the replisome remains Author contributions: H.A.J., B.J.S., C.T.C., and J.C. designed research; H.A.J., B.J.S., C.T.C., bound to the arresting lesion, it may sterically obstruct repair or and J.C. performed research; H.A.J., B.J.S., C.T.C., and J.C. analyzed data; and H.A.J. wrote bypass from occurring. Conversely, complete dissociation of the the paper. replisome would likely abolish the licensing for the replication The authors declare no conflict of interest. fork and expose DNA ends that have the potential to recombine, This article is a PNAS Direct Submission. generating deletions, duplications, or rearrangements on the 1To whom correspondence should be addressed. E-mail: [email protected]. chromosome. Recent studies in vitro have suggested that dynamic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. interactions between replisome components may play a role in 1073/pnas.1300624110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300624110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A B β are required to maintain DNA polymerase binding to the DNA template (19, 20). The processing intermediates were induced Polymerase and Clamp WT specifically by the disruption of the DNA polymerase III sub- Helicase- dnaX dnaB Primase C units, because no processing intermediates were induced in wild- dnaG Complex 30 dnaN type cells at the restrictive temperature or following inactivation of the nonessential proofreading subunit, e (DnaQ) (Fig. 2 and 6 dnaE dnaQ Fig. S1). Similar to the DNA polymerase subunits, UV-like processing DnaB WT intermediates were also induced following inactivation of τ DnaG dnaX dnaB (DnaX), which is responsible for coupling the polymerases to the C replisome, coordinating leading and lagging strand synthesis, and Coupling- 42 dnaN dnaG clamp loading repetitive cycling of the processivity factor onto the lagging complex dnaE dnaQ strand template (21–25). The observed induction of UV-like C intermediates following inactivation of the DNA polymerase or UV Irrad Shift Temperature to 42 C coupling factor indicates that the UV-induced processing of replication forks is likely to involve the transient dissociation of 1.0 the polymerase from the DNA. dnaQ WT WT The helicase–primase complex interacts with τ and the holo- Post-UV Post-TS dnaG 0.1 enzyme and tracks along the lagging strand template, serving to dnaE – α β dnaN unwind and prime it during replication (26 28). In contrast to , , dnaX τ Rate of DNA Synthesis dnaB or , inactivation of either the helicase (DnaB) or primase (DnaG) -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 -20 0 20 40 60 80 led to the production of aberrant structural intermediates that Minutes Post Treatment were unlike any of those that are observed during the pro- Fig. 1. Replication is disrupted by UV-induced damage or following inac- cessing of UV-induced damage (Fig. 2 and Fig. S1), arguing that tivation of the DNA polymerase, τ complex, or helicase–primase complex. (A) disruption of the helicase complex does not normally occur A diagram of the replisome, indicating the subunits of each protein complex. during the recovery of replication after UV-induced arrest. (B) Thermosensitive mutants that inactivate the polymerase core, τ complex, or helicase complex are viable at 30 °C but fail to grow at the restrictive
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]
  • Restoration of Mir-193A Expression Is Tumor-Suppressive in MYC
    Bharambe et al. Acta Neuropathologica Communications (2020) 8:70 https://doi.org/10.1186/s40478-020-00942-5 RESEARCH Open Access Restoration of miR-193a expression is tumor-suppressive in MYC amplified Group 3 medulloblastoma Harish Shrikrishna Bharambe1,2, Annada Joshi1, Kedar Yogi1,2, Sadaf Kazi1 and Neelam Vishwanath Shirsat1,2* Abstract Medulloblastoma, a highly malignant pediatric brain tumor, consists of four molecular subgroups, namely WNT, SHH, Group 3, and Group 4. The expression of miR-193a, a WNT subgroup-specific microRNA, was found to be induced by MYC, an oncogenic target of the canonical WNT signaling. MiR-193a is not expressed in Group 3 medulloblastomas, despite MYC expression, as a result of promoter hypermethylation. Restoration of miR-193a expression in the MYC amplified Group 3 medulloblastoma cells resulted in inhibition of growth, tumorigenicity, and an increase in radiation sensitivity. MAX,STMN1, and DCAF7 were identified as novel targets of miR-193a. MiR- 193a mediated downregulation of MAX could suppress MYC activity since it is an obligate hetero-dimerization partner of MYC. MYC induced expression of miR-193a, therefore, seems to act as a feedback inhibitor of MYC signaling. The expression of miR-193a resulted in widespread repression of gene expression that included not only several cell cycle regulators, WNT, NOTCH signaling genes, and those encoding DNA replication machinery, but also several chromatin modifiers like SWI/SNF family genes and histone-encoding genes. MiR-193a expression brought about a reduction in the global levels of H3K4me3, H3K27ac, the histone marks of active chromatin, and an increase in the levels of H3K27me3, a repressive chromatin mark.
    [Show full text]
  • Rnase-H-Based Assays Utilizing Modified Rna
    (19) TZZ ¥_T (11) EP 2 279 263 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) 04.09.2013 Bulletin 2013/36 (86) International application number: (21) Application number: 09739895.2 PCT/US2009/042454 (22) Date of filing: 30.04.2009 (87) International publication number: WO 2009/135093 (05.11.2009 Gazette 2009/45) (54) RNASE-H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS TESTS AUF RNASE-H-BASIS MIT MODIFIZIERTEN RNA-MONOMEREN DOSAGES À BASE DE RNASE-H UTILISANT DES MONOMÈRES D’ARN MODIFIÉS (84) Designated Contracting States: • ROSE, Scott AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Coralville, IA 52241 (US) HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL • DOBOSY, Joseph PT RO SE SI SK TR Coralville, IA 52241 (US) (30) Priority: 30.04.2008 US 49204 P (74) Representative: Grünecker, Kinkeldey, Stockmair & Schwanhäusser (43) Date of publication of application: Leopoldstrasse 4 02.02.2011 Bulletin 2011/05 80802 München (DE) (60) Divisional application: (56) References cited: 13173388.3 EP-A- 1 367 136 WO-A-01/21813 13173389.1 WO-A-03/074724 WO-A-2004/059012 13173390.9 WO-A-2007/062495 WO-A2-2005/021776 13173391.7 WO-A2-2007/141580 US-A- 5 744 308 US-A- 5 830 664 (73) Proprietor: Integrated Dna Technologies, Inc. Coralville, IA 52241 (US) • ITAYA M ET AL: "Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme (72) Inventors: thermophile Thermus thermophilus HB8: a • WALDER, Joseph, Alan thermostable RNase H can functionally replace Chicago, IL 60645 (US) the Escherichia coli enzyme in vivo." NUCLEIC • BEHLKE, Mark, Aaron ACIDS RESEARCH 25 AUG 1991, vol.
    [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]
  • Telomerase Reverse Transcriptase Expression Elevated by Avian Leukosis Virus Integration in B Cell Lymphomas
    Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas Feng Yang, Rena R. Xian*, Yingying Li, Tatjana S. Polony, and Karen L. Beemon† Department of Biology, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218 Communicated by Saul Roseman, The Johns Hopkins University, Baltimore, MD, September 26, 2007 (received for review May 11, 2007) Simple retroviruses induce tumors by integrating into the host integration sites are believed to result from strong biological genome, activating cellular oncogenes and microRNAs, or inacti- selection (7). vating tumor suppressor genes. The identification of these genes Avian leukosis virus (ALV) is a simple retrovirus that does not elucidates molecular mechanisms of tumorigenesis. In this study, encode an oncogene but that can induce tumors by integrating we identified avian leukosis virus (ALV) proviral integration sites in near oncogenes, introducing a strong promoter or enhancer rapid-onset B cell lymphomas arising <12 weeks after infection of sequence (8–12). Typically, ALV induces B cell lymphomas to chicken embryos. By using inverse PCR, 28 unique viral integration develop in Ϸ6 months and frequently involves proviral integra- sites were identified in rapid-onset tumors. Integrations in the tions, resulting in deregulation of the cellular transcription telomerase reverse transcriptase (TERT) promoter/enhancer region factor, myc, as well as bic (precursor to microRNA 155) (10–12). were observed in four different tumors, suggesting that this is a In addition, B cell lymphomas with a more rapid onset (lethal in common integration site. These provirus integrations ranged from Ͻ3 months) have been observed with clonal proviral insertions 217 to 2,584 bp upstream of the TERT transcription initiation site into the myb gene locus, resulting in overexpression of a trun- and were all in the opposite transcriptional orientation to TERT.
    [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]
  • Hexameric Helicase G40P Unwinds DNA in Single Base Pair Steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7*
    RESEARCH ARTICLE Hexameric helicase G40P unwinds DNA in single base pair steps Michael Schlierf1,2*, Ganggang Wang3, Xiaojiang S Chen3, Taekjip Ha1,4,5,6,7* 1Physics Department and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Illinois, United States; 2B CUBE – Center for Molecular Bioengineering, Technische Universita¨ t Dresden, Dresden, Germany; 3Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, United States; 4Howard Hughes Medical Institute, Baltimore, United States; 5Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, United States; 6Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States; 7Department of Biophysics, Johns Hopkins University, Baltimore, United States Abstract Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPgS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.
    [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]