Supplemental Table 7. Every Significant Association
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DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli
DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kath, James Evon. 2016. DNA Polymerase Exchange and Lesion Bypass in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718716 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ! ! ! ! ! ! ! DNA!polymerase!exchange!and!lesion!bypass!in!Escherichia)coli! ! A!dissertation!presented! by! James!Evon!Kath! to! The!Committee!on!Higher!Degrees!in!Biophysics! ! in!partial!fulfillment!of!the!requirements! for!the!degree!of! Doctor!of!Philosophy! in!the!subject!of! Biophysics! ! Harvard!University! Cambridge,!Massachusetts! October!2015! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ©!2015!L!James!E.!Kath.!Some!Rights!Reserved.! ! This!work!is!licensed!under!the!Creative!Commons!Attribution!3.0!United!States!License.!To! view!a!copy!of!this!license,!visit:!http://creativecommons.org/licenses/By/3.0/us! ! ! Dissertation!Advisor:!Professor!Joseph!J.!Loparo! ! ! !!!!!!!!James!Evon!Kath! ! DNA$polymerase$exchange$and$lesion$bypass$in$Escherichia)coli$ $ Abstract$ ! Translesion! synthesis! (TLS)! alleviates! -
Ribonuclease A: Disulfide Bonds, Conformational Stability, and Cytotoxicity
Ribonuclease A: Disulfide Bonds, Conformational Stability, and Cytotoxicity by Tony A. Klink A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 2()()() r OJ A dissertation entitled Ribonuclease A: Disulfide Bonds, Conformational Stability and Cytotoxicity submitted to the Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Tony Anthony Klink Date of Final Oral Examination: August 4, 2000 Month & Year Degree to be awarded: December May August 2000 • * * * * * * • * * • * • • • • • * • • • * • • • • • • • • • • • • • * * • * • * * * • * • * • • • • • • * App oval Signature. f Dissertation Readers: Signature, Dean of Graduate School ---..., v.C~vJ,1A. 5. lJ,~k;at 1 Abstract Disulfide bonds between the side chains of cysteine residues are the only common cross links in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-CysllO, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the confonnational stability and catalytic activity of RNase A was detennined using variants in which each cystine was replaced independently with a pair of alanine residues. Of the four disulfide bonds, the Cys40-Cys95 and Cys65-Cys72 cross-links are the least important to confonnational stability. Removing these disulfide bonds leads to RNase A variants that have Tm values below that of the wild-type enzyme but above physiological temperature. Unlike wild-type RNase A. G88R RNase A is toxic to cancer cells. To investigate the relationship between conformational stability and cytotoxicity, the C40AlC95A and C65A1C72A variants were made in the G88R background. -
Phylogenetic Analysis Reveals an Ancient Gene Duplication As The
1 Phylogenetic analysis reveals an ancient gene duplication as 2 the origin of the MdtABC efflux pump. 3 4 Kamil Górecki1, Megan M. McEvoy1,2,3 5 1Institute for Society & Genetics, 2Department of MicroBiology, Immunology & Molecular 6 Genetics, and 3Molecular Biology Institute, University of California, Los Angeles, CA 90095, 7 United States of America 8 Corresponding author: [email protected] (M.M.M.) 9 1 10 Abstract 11 The efflux pumps from the Resistance-Nodulation-Division family, RND, are main 12 contributors to intrinsic antibiotic resistance in Gram-negative bacteria. Among this family, the 13 MdtABC pump is unusual by having two inner membrane components. The two components, 14 MdtB and MdtC are homologs, therefore it is evident that the two components arose by gene 15 duplication. In this paper, we describe the results obtained from a phylogenetic analysis of the 16 MdtBC pumps in the context of other RNDs. We show that the individual inner membrane 17 components (MdtB and MdtC) are conserved throughout the Proteobacterial species and that their 18 existence is a result of a single gene duplication. We argue that this gene duplication was an ancient 19 event which occurred before the split of Proteobacteria into Alpha-, Beta- and Gamma- classes. 20 Moreover, we find that the MdtABC pumps and the MexMN pump from Pseudomonas aeruginosa 21 share a close common ancestor, suggesting the MexMN pump arose by another gene duplication 22 event of the original Mdt ancestor. Taken together, these results shed light on the evolution of the 23 RND efflux pumps and demonstrate the ancient origin of the Mdt pumps and suggest that the core 24 bacterial efflux pump repertoires have been generally stable throughout the course of evolution. -
Analysis of Carbohydrates and Glycoconjugates by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: an Update for 2011–2012
ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2011–2012 David J. Harvey* Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford OX1 3QU, UK Received 19 June 2014; accepted 15 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21471 This review is the seventh update of the original article published text. As the review is designed to complement the earlier work, in 1999 on the application of MALDI mass spectrometry to the structural formulae, etc. that were presented earlier are not analysis of carbohydrates and glycoconjugates and brings repeated. However, a citation to the structure in the earlier work coverage of the literature to the end of 2012. General aspects is indicated by its number with a prefix (i.e., 1/x refers to such as theory of the MALDI process, matrices, derivatization, structure x in the first review and 2/x to structure x the second). MALDI imaging, and fragmentation are covered in the first part Books, general reviews, and review-type articles directly of the review and applications to various structural types concerned with, or including, MALDI analysis of carbohydrates constitute the remainder. The main groups of compound are and glycoconjugates to have been published during the review oligo- and poly-saccharides, glycoproteins, glycolipids, glyco- period are listed in Table 1. Reviews relating to more specific sides, and biopharmaceuticals. Much of this material is presented aspects of MALDI analysis are listed in the appropriate sections. in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and II. -
Determination of Pk, Values of the Histidine Side
Protein Science (1997), 6:1937-1944. Cambridge University Press. Printed in the USA Copyright 0 1997 The Protein Society Determination of pK, values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis TUN LIU, MARGRET RYAN, FREDERICK W. DAHLQUIST, AND 0. HAYES GRIFFITH Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 (RECEIVEDDecember 4, 1996: ACCEPTEDMay 19, 1997) Abstract Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pK,, values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selec- tively labeled with '3C"-histidine. A series of 'H-I3C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 'H-"C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pK,, values of 7.6 and 6.9, respectively. His92 and His227 exhibit pK, values of 5.4 and 6.9. -
Comparative Genomics and Functional Annotation of Bacterial Transporters
Physics of Life Reviews 5 (2008) 22–49 www.elsevier.com/locate/plrev Review Comparative genomics and functional annotation of bacterial transporters Mikhail S. Gelfand a,b,∗, Dmitry A. Rodionov a,c a Institute for Information Transmission Problems, Russian Academy of Sciences, Bolshoi Karetny pereulok 19, Moscow 127994, Russia b Department of Bioengineering and Bioinformatics, Moscow State University, Russia c Burnham Institute for Medical Research, La Jolla, CA 92037, USA Received 6 October 2007; received in revised form 8 October 2007; accepted 10 October 2007 Available online 24 October 2007 Communicated by M. Frank-Kamenetskii Abstract Transport proteins are difficult to study experimentally, and because of that their functional characterization trails that of en- zymes. The comparative genomic analysis is a powerful approach to functional annotation of proteins, which makes it possible to utilize the genomic sequence data from thousands of organisms. The use of computational techniques allows one to identify candidate transporters, predict their structure and localization in the membrane, and perform detailed functional annotation, which includes substrate specificity and cellular role. We overview the main techniques of analysis of transporters’ structure and function. We consider the most popular algorithms to identify transmembrane segments in protein sequences and to predict topology of multispanning proteins. We describe the main approaches of the comparative genomics, and how they may be applied to the analysis of transporters, and provide examples showing how combinations of these techniques is used for functional annotation of new transporter specificities in known families, characterization of new families, and prediction of novel transport mechanisms. © 2007 Elsevier B.V. -
United States Patent (19) 11 Patent Number: 5,981,835 Austin-Phillips Et Al
USOO598.1835A United States Patent (19) 11 Patent Number: 5,981,835 Austin-Phillips et al. (45) Date of Patent: Nov. 9, 1999 54) TRANSGENIC PLANTS AS AN Brown and Atanassov (1985), Role of genetic background in ALTERNATIVE SOURCE OF Somatic embryogenesis in Medicago. Plant Cell Tissue LIGNOCELLULOSC-DEGRADING Organ Culture 4:107-114. ENZYMES Carrer et al. (1993), Kanamycin resistance as a Selectable marker for plastid transformation in tobacco. Mol. Gen. 75 Inventors: Sandra Austin-Phillips; Richard R. Genet. 241:49-56. Burgess, both of Madison; Thomas L. Castillo et al. (1994), Rapid production of fertile transgenic German, Hollandale; Thomas plants of Rye. Bio/Technology 12:1366–1371. Ziegelhoffer, Madison, all of Wis. Comai et al. (1990), Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS 73 Assignee: Wisconsin Alumni Research elements. Plant Mol. Biol. 15:373-381. Foundation, Madison, Wis. Coughlan, M.P. (1988), Staining Techniques for the Detec tion of the Individual Components of Cellulolytic Enzyme 21 Appl. No.: 08/883,495 Systems. Methods in Enzymology 160:135-144. de Castro Silva Filho et al. (1996), Mitochondrial and 22 Filed: Jun. 26, 1997 chloroplast targeting Sequences in tandem modify protein import specificity in plant organelles. Plant Mol. Biol. Related U.S. Application Data 30:769-78O. 60 Provisional application No. 60/028,718, Oct. 17, 1996. Divne et al. (1994), The three-dimensional crystal structure 51 Int. Cl. ............................. C12N 15/82; C12N 5/04; of the catalytic core of cellobiohydrolase I from Tricho AO1H 5/00 derma reesei. Science 265:524-528. -
The Protein Import Machinery of Mitochondria-A Regulatory Hub In
Cell Metabolism Perspective The Protein Import Machinery of Mitochondria—A Regulatory Hub in Metabolism, Stress, and Disease Angelika B. Harbauer,1,2,3,4 Rene´ P. Zahedi,5 Albert Sickmann,5,6 Nikolaus Pfanner,1,4,* and Chris Meisinger1,4,* 1Institut fu¨ r Biochemie und Molekularbiologie, ZBMZ 2Trinationales Graduiertenkolleg 1478 3Faculty of Biology 4BIOSS Centre for Biological Signalling Studies Universita¨ t Freiburg, 79104 Freiburg, Germany 5Leibniz-Institute for Analytical Sciences–ISAS–e.V., 44139 Dortmund, Germany 6Medizinisches Proteom-Center, Ruhr-Universita¨ t Bochum, 44801 Bochum, Germany *Correspondence: [email protected] (N.P.), [email protected] (C.M.) http://dx.doi.org/10.1016/j.cmet.2014.01.010 Mitochondria fulfill central functions in bioenergetics, metabolism, and apoptosis. They import more than 1,000 different proteins from the cytosol. It had been assumed that the protein import machinery is constitu- tively active and not subject to detailed regulation. However, recent studies indicate that mitochondrial protein import is regulated at multiple levels connected to cellular metabolism, signaling, stress, and patho- genesis of diseases. Here, we discuss the molecular mechanisms of import regulation and their implications for mitochondrial homeostasis. The protein import activity can function as a sensor of mitochondrial fitness and provides a direct means of regulating biogenesis, composition, and turnover of the organelle. Introduction machinery is essential for the viability -
DNA Polymerase V Activity Is Autoregulated by a Novel Intrinsic DNA-Dependent
1 2 DNA polymerase V activity is autoregulated by a novel intrinsic DNA-dependent 3 ATPase 4 Aysen L. Erdem1, Malgorzata Jaszczur1, Jeffrey G. Bertram1, Roger Woodgate2, Michael M. Cox3 & 5 Myron F. Goodman1 6 1Departments of Biological Sciences and Chemistry, University of Southern California, University 7 Park, Los Angeles, California 90089-2910, USA. 2Laboratory of Genomic Integrity, National 8 Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 9 Maryland 20892-3371, USA. 3Department of Biochemistry, University of Wisconsin-Madison, 10 Madison, Wisconsin 53706, USA. 11 12 Escherichia coli DNA polymerase V (pol V), a heterotrimeric complex composed of UmuD′2C, 13 is marginally active. ATP and RecA play essential roles in the activation of pol V for DNA 14 synthesis including translesion synthesis (TLS). We have established three features of the roles 15 of ATP and RecA. 1) RecA-activated DNA polymerase V (pol V Mut), is a DNA-dependent 16 ATPase; 2) bound ATP is required for DNA synthesis; 3) pol V Mut function is regulated by 17 ATP, with ATP required to bind primer/template (p/t) DNA and ATP hydrolysis triggering 18 dissociation from the DNA. Pol V Mut formed with an ATPase-deficient RecA E38K/K72R 19 mutant hydrolyzes ATP rapidly, establishing the DNA-dependent ATPase as an intrinsic 20 property of pol V Mut distinct from the ATP hydrolytic activity of RecA when bound to 21 single-stranded (ss)DNA as a nucleoprotein filament (RecA*). No similar ATPase activity or 22 autoregulatory mechanism has previously been found for a DNA polymerase. -
Regulation of Gene Expression by the Camp-Crp System in the Soil Bacterium Pseudomonas Putida
UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR Regulation of gene expression by the cAMP-Crp system in the soil bacterium Pseudomonas putida TESIS DOCTORAL Memoria presentada para optar al grado de Doctor en Ciencias Alejandro Arce Rodríguez DIRECTORES DE TESIS: Víctor de Lorenzo Prieto Belén Calles Arenales CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (CSIC) CENTRO NACIONAL DE BIOTECNOLOGÍA Madrid, 2012 En memoria de mi abuela Alicia Corrales Campos (1938-2012), quien un día se durmió en su particular “País de las maravillas” para no volver jamás. QEPD A Dios y a mi familia. Soy quien soy gracias a ustedes! In memory of my grandmother Alicia Corrales Campos (1938-2012), who one day fell asleep in her particular “Wonderland” to never come back. RIP To God and my family. Acknowledgements This work would not have been possible without the support of many people who I would like to thank with a few words. To Victor de Lorenzo, to whom I am especially grateful for the opportunity to join his group, for his teaching, his guidance and specially for supporting and encouraging me always, in the bad and good moments. Thank you very much F !! Also to Beléeeeeeen Calles for accepting to be the co-director of this Thesis, and of course for her teaching, her patience, her support and her friendship. Thank you both for showing me how to be a better scientist! Many thanks to Fernando Rojo, for accepting to be my tutor. I would like to thank Tino Krell and Raúl Platero, for their contribution with the ITC experiments and for the useful discussions about the thermodynamic properties of Crp. -
Sequence Homology Between Purple Acid Phosphatases And
Volume 263, number 2, 265-268 FEBS 08346 April 1990 Sequence homology between purple acid phosphatases and phosphoprotein phosphatases Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers? John B. Vincent and Bruce A. Averill University of Virginia, Department of Chemistry, Charlottesville, VA 22901, USA Received 12 January 1990; revised version received 26 February 1990 The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal-binding residues. The phosphoprotein phosphatases I, 2A and 2B are proposed to be iron-zinc metalloenzymeswith active sites isostructural (or nearly so) with those of the purple phosphatases, Protein phosphatase; Purple acid phosphatase; Sequence homology 1. INTRODUCTION tain fungi and Drosophila have been shown to be highly homologous (50-90°70) to those of mammalian PP1 Phosphoprotein phosphatases (PPs) are a class of and PP2A [13,16-20], suggesting that the phosphopro- mammalian regulatory enzymes that catalyze the tein phosphatases may be widely distributed. dephosphorylation of phosphoserine and phospho- Mammalian purple acid phosphatases (PAPs) are threonine proteins [1,2]. Phosphoprotein phosphatase novel enzymes of molecular mass -37 kDa that contain 1 (PP1), which is inhibited by inhibitor-1 and -2, an oxide-bridged dinuclear iron active site. The amino generally occurs in a glycogen- or myosin-bound form. acid sequences of the enzymes from bovine spleen, por- The type 2 enzymes (insensitive to the above inhibitors) cine uterine fluid, and human placenta are highly are further subdivided into three classes. PP2A is a homologous (-90o70), again indicating a close relation- cytosolic enzyme that possesses broad reactivity, while ship [21,22]. -
Reca Acts As a Switch to Regulate Polymerase Occupancy in a Moving Replication Fork
RecA acts as a switch to regulate polymerase occupancy in a moving replication fork Chiara Indiania,1, Meghna Patelb, Myron F. Goodmanb, and Mike E. O’Donnellc,1 aManhattan College, Riverdale, NY 10471; bDepartment of Biological Sciences, University of Southern California, Los Angeles, CA 90089; and cHoward Hughes Medical Institute, Rockefeller University, New York, NY 10065 Contributed by Mike O’Donnell, February 19, 2013 (sent for review February 6, 2013) This report discovers a role of Escherichia coli RecA, the cellular regulates DNA polymerase access to the replication fork. Sur- recombinase, in directing the action of several DNA polymerases prisingly, RecA strongly inhibits fork progression by Pol III repli- at the replication fork. Bulk chromosome replication is performed somes, yet RecA stimulates TLS Pol II and Pol IV replisomes. The by DNA polymerase (Pol) III. However, E. coli contains translesion mechanism that underlies the opposite effects of RecA on Pol III synthesis (TLS) Pols II, IV, and V that also function with the helicase, and the TLS Pol replisomes involves single-strand binding protein primase, and sliding clamp in the replisome. Surprisingly, we find (SSB). Although all of the polymerases function with SSB on the that RecA specifically activates replisomes that contain TLS Pols. In template strand, SSB inhibits TLS Pol function in the context of sharp contrast, RecA severely inhibits the Pol III replisome. Given a replisome, presumably acting in trans by binding lagging-strand the opposite effects of RecA on Pol III and TLS replisomes, we pro- ssDNA. RecA relieves the SSB induced repression of TLS Pol pose that RecA acts as a switch to regulate the occupancy of poly- replisomes and stimulates their action, while inhibiting the Pol III merases within a moving replisome.