DOCSLIB.ORG

Dynamics of Α-Helical Subdomain Rotation in the Intact Maltose ATP-Binding Cassette Transporter

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Dynamics of Α-Helical Subdomain Rotation in the Intact Maltose ATP-Binding Cassette Transporter Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter Cédric Orellea, Frances Joan D. Alvareza, Michael L. Oldhamb, Arnaud Orellea, Theodore E. Wileya, Jue Chenb, and Amy L. Davidsona,1 aChemistry Department, and bHoward Hughes Medical Institute, Purdue University, 560 Oval Dr, West Lafayette, IN 47906 Edited by Christopher Miller, Brandeis University, Waltham, MA, and approved October 4, 2010 (received for review May 12, 2010) ATP-binding cassette (ABC) transporters are powered by a nucleo- events, a ∼10° rotation of the α-helical subdomains relative to tide-binding domain dimer that opens and closes during cycles of the RecA-like subdomains and a ∼14° rotation of the RecA-like ATP hydrolysis. These domains consist of a RecA-like subdomain subdomains relative to the C-terminal regulatory domains (23). and an α-helical subdomain that is specific to the family. Many Analysis of the helical subdomain rotation is crucial for a detailed studies on isolated domains suggest that the helical subdomain ro- understanding of how ATP hydrolysis is stimulated by MBP and tates toward the RecA-like subdomain in response to ATP binding, coupled to transport. We therefore determined the requirements moving the family signature motif into a favorable position to in- for subdomain rotation and defined the sequence of rotational teract with the nucleotide across the dimer interface. Moreover, events in the catalytic cycle. the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the Results rotation in interdomain communication. Electron paramagnetic Positioning Spin Labels to Detect α-Helical Subdomain Rotation in resonance spectroscopy was used to study the dynamics of this MalK. The maltose transporter has been crystallized in an inward- Escherichia coli rotation in the intact maltose transporter MalFGK2. facing conformation in the absence of nucleotide (23), and an This importer requires a periplasmic maltose-binding protein (MBP) outward-facing conformational intermediate, stabilized by bind- that activates ATP hydrolysis by promoting the closure of the ing of both ATP and MBP, that may resemble the transition state cassette dimer (MalK2). Whereas this rotation occurred during the (22, 24, 25) (Fig. 1A). Alignment of the RecA-like subdomains of BIOCHEMISTRY transport cycle, it required not only trinucleotide, but also MBP, these two structures (Fig. 1B) reveals an inward rotation of the suggesting it is part of a global conformational change in the trans- helical subdomain toward the RecA-like subdomain, as seen in 2þ porter. Interaction of AMP-PNP-Mg and a MBP that is locked in many isolated NBD structures upon ATP binding (5–8). These a closed conformation induced a transition from open MalK2 to two subdomains are connected by the Q-loop (27) that lines part semiopen MalK2 without significant subdomain rotation. Inward of the cleft surrounding the coupling helix of the transmembrane rotation of the helical subdomain and complete closure of MalK2 domains MalF and MalG. The Q-loop is expected to undergo therefore appear to be coupled to the reorientation of transmem- conformational changes upon subdomain rotation that might brane helices and the opening of MBP, events that promote trans- be detected as a change in mobility of an attached spin label fer of maltose into the transporter. After ATP hydrolysis, the helical (SL). Residues 83–87 in the Q-loop were individually mutated subdomain rotates out as MalK2 opens, resetting the transporter in to cysteine. Residues in the helical subdomain (residues V120, an inward-facing conformation. Q122, and A124) and in the RecA-line subdomain (residues A167, L168, and Q171) (Fig. 1B) were also mutated to cysteines, EPR spectroscopy ∣ transport mechanism ∣ membrane protein individually and in pairs, to measure changes in the distance between these two subdomains upon rotation. Residues were TP-binding cassette (ABC) transporters belong to one of the selected for mutation based on distance constraints (28, 29) Alargest protein superfamilies in organisms, and mediate the and side-chain solvent-accessibility [http://mobyle.rpbs.univ- translocation of a wide range of substrates across the membrane paris-diderot.fr/cgi-bin/portal.py?form=ASA (30)], to maximize (1). These transporters typically contain two transmembrane do- spin-labeling efficiency. mains (TMDs) and are energized by a nucleotide-binding domain Plasmids carrying mutation(s) encoding the cysteine substitu- (NBD) dimer that closes and opens during cycles of ATP binding tions in the malK gene were used to transform an E. coli strain and hydrolysis (2, 3). Each NBD consists of a RecA-like subdo- containing a chromosomal deletion of malK, and were tested main, found in numerous ATPases (4), and an α-helical subdo- for function by complementation, as judged by the red color main that is specific to the ABC family (5). Crystallographic of the colonies on maltose MacConkey agar plates (22). By this studies (5–8) and molecular dynamic simulations (9, 10), criterion, all of the substitutions in the helical and RecA-like sub- performed on isolated NBDs, suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP domains were functional, but only one of the Q-loop substitutions binding. This rotation positions the ABC family signature motif (S83C) was functional. The mutants were purified, spin-labeled, to interact with nucleotide across the dimer interface so that the and tested for MBP-stimulated ATPase activity (Table S1). All NBDs can close to hydrolyze ATP. After ATP hydrolysis, it is of the double mutants showed a strong stimulation by MBP,which suggested that the helical subdomain rotates away (5, 11). In is characteristic of the maltose transporter (21). However, only addition, the TMDs contact the NBDs at the interface between four of the double mutants, those that retained reasonably high these subdomains (12–15), suggesting a putative role of the rota- tion in interdomain communication (16). Here, we used site- Author contributions: C.O. and A.L.D. designed research; C.O., F.J.D.A., M.L.O., A.O., and directed spin labeling electron paramagnetic resonance (EPR) T.E.W. performed research; F.J.D.A., M.L.O., and J.C. contributed new reagents/analytic spectroscopy (17, 18) to study the dynamics of this rotation in tools; C.O., J.C., and A.L.D. analyzed data; and C.O. and A.L.D. wrote the paper. the intact Escherichia coli maltose transporter MalFGK2 (19, 20). The authors declare no conflict of interest. A periplasmic maltose-binding protein (MBP) delivers the sub- This article is a PNAS Direct Submission. strate to the transporter and stimulates its ATPase activity (21). 1To whom correspondence should be addressed. E-mail: [email protected]. In the maltose transporter, closure of the NBD dimer requires This article contains supporting information online at www.pnas.org/lookup/suppl/ both ATP and MBP (22) and involves two separate rotational doi:10.1073/pnas.1006544107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006544107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 (ADP-Mg2þ-bound) state (22), increased the mobility of S83C- SL relative to the catalytic intermediate, though the spectrum was not superimposable with that of the apo state (Fig. 2). Changes in mobility of SL positioned at V120C and Q122C in the helical subdomain were also seen only following the addition of both AMP-PNP-Mg2þ and maltose-MBP (Fig. 2). In contrast, changes in mobility of SL positioned at L168C and, to a lesser extent, Q171C in the RecA-like subdomain (Fig. S1) are seen upon ad- dition of AMP-PNP-Mg2þ only, in addition to the more substan- tial changes apparent when both ligands are present. Crystal structures of isolated NBDs indicate that when the NBDs are open, nucleotide binds to the RecA-like subdomain rather than the helical subdomain (31) consistent with the observation that Fig. 1. Structural views of the maltose ABC transporter. (A) Structures of the SLs positioned in the RecA-like subdomain, but not the helical maltose transporter in apo (Left, PDB 3FH6) and catalytic intermediate (Right, subdomain, sensed nucleotide binding. The altered mobilities of PDB 2R6G) states. The transmembrane domains MalF and MalG are both SLs in the helical subdomain in the catalytic intermediate may shown in orange. MBP is in blue. The RecA-like subdomain (gray), α-helical reflect helical subdomain rotation, which brings the SL side subdomain (red), and regulatory domain (purple) of MalK are distinguished. chains closer to residues in the RecA-like subdomain. ATP (green) and maltose (cyan) molecules are present only in the catalytic intermediate. (B) Closeup of the MalK/MalG interface. Structures of the apo and catalytic intermediate state are superimposed based on the RecA- EPR Analysis of Doubly Labeled Mutants in the Apo State. To demon- like subdomain to visualize the helical subdomain rotation and movement strate the rotation of the helical subdomain relative to the RecA- of the coupling helix. MalG is rendered in yellow (apo state) and orange (cat- like subdomain, we tested several different pairs of spin-labels to alytic intermediate state). The coupling helix (or EAA loop) of MalG is shown determine the distance between these subdomains (Table S2). as a cylinder. The RecA-like and helical subdomains of MalK (colored as in A) When the interspin distance is >25 Å, the spectrum of a doubly from the apo and catalytic intermediate states are shown in lighter and dar- labeled protein in continuous wave (cw)-EPR strictly corresponds ker hues, respectively (figure modified from ref. 23). Residue Q82 defining to the sum of the spectra of the noninteracting single mutants. the Q-loop (blue) is shown as stick for the catalytic intermediate state. Interspin distances can be determined at room temperature in Spheres indicate the positions of residues spin-labeled in this study (Q122 – and A167 are indicated).
Load more

Recommended publications
  • Examining the Mechanism of a Heavy- Metal ABC Exporter Dennis Hicks University of San Francisco, [email protected]
    The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Spring 3-31-2019 NaAtm1: Examining the mechanism of a Heavy- metal ABC Exporter Dennis Hicks University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biochemistry Commons Recommended Citation Hicks, Dennis, "NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter" (2019). Master's Theses. 1204. https://repository.usfca.edu/thes/1204 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter A thesis presented to the faculty of the Department of Chemistry at the University of San Francisco in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Written by Dennis Hicks Bachelor of Science in Biochemistry San Francisco State University August 2019 NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter Thesis written by Dennis Hicks This thesis is written under the guidance of the Faculty Advisory Committee, and approved by all its members, has been accepted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at the University of San Francisco Thesis Committee Janet G.
    [Show full text]
  • Structure and Mechanism of a Group-I Cobalt Energy Coupling Factor Transporter
    Cell Research (2017) 27:675-687. © 2017 IBCB, SIBS, CAS All rights reserved 1001-0602/17 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr Structure and mechanism of a group-I cobalt energy coupling factor transporter Zhihao Bao1, 2, 3, *, Xiaofeng Qi1, 3, *, Sen Hong1, 3, Ke Xu1, 3, Fangyuan He1, Minhua Zhang1, Jiugeng Chen1, Daiyin Chao1, Wei Zhao4, Dianfan Li4, Jiawei Wang5, Peng Zhang1 1National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 2Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 3University of Chinese Academy of Sciences, Beijing 100049, China; 4State Key Laboratory of Molecular Biology, National Center for Protein Sciences, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chi- nese Academy of Sciences, Shanghai 200031, China; 5State Key Laboratory of Membrane Biology, School of Life Sciences, Tsing- hua University, Beijing 100084, China Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently iden- tified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are mod- ular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A’. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transport- ers is very limited, partly due to a lack of transporter complex structural information.
    [Show full text]
  • Reca and DNA Recombination: a Review of Molecular Mechanisms
    Biochemical Society Transactions (2019) 47 1511–1531 https://doi.org/10.1042/BST20190558 Review Article RecA and DNA recombination: a review of molecular mechanisms Downloaded from https://portlandpress.com/biochemsoctrans/article-pdf/47/5/1511/859295/bst-2019-0558.pdf by Tata Institute of Fundamental Research user on 20 January 2020 Elsa del Val1,2, William Nasser1, Hafid Abaibou2 and Sylvie Reverchon1 1Microbiologie, Adaptation et Pathogénie, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, 11 Avenue Jean Capelle, F-69621 Villeurbanne, France; 2Molecular Innovation Unit, Centre Christophe Mérieux, BioMérieux, 5 rue des Berges, 38024 Grenoble Cedex 01, France Correspondence: Sylvie Reverchon ([email protected]) or Hafid Abaibou ([email protected]) Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combin- ation of DNA synthesis, ligation and resolution.
    [Show full text]
  • The Universal Stress Proteins of Bacteria
    The Universal Stress Proteins of Bacteria Dominic Bradley Department of Life Sciences A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Imperial College London The Universal Stress Proteins of Bacteria Declaration of Originality I, Dominic Bradley, declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institute of tertiary education. Information derived from the published and unpublished work of others has been acknowledged in the text and a list of references is given in the bibliography. 1 The Universal Stress Proteins of Bacteria Abstract Universal stress proteins (USPs) are a widespread and abundant protein family often linked to survival during stress. However, their exact biochemical and cellular roles are incompletely understood. Mycobacterium tuberculosis (Mtb) has 10 USPs, of which Rv1636 appears to be unique in its domain structure and being the only USP conserved in M. leprae. Over-expression of Rv1636 in M. smegmatis indicated that this protein does not share the growth arrest phenotype of another Mtb USP, Rv2623, suggesting distinct roles for the Mtb USPs. Purified Rv1636 was shown to have novel nucleotide binding capabilities when subjected to UV crosslinking. A range of site-directed mutants of Rv1636 were produced, including mutations within a predicted nucleotide binding motif, with the aim of identifying and characterising key residues within the Rv1636 protein. Further putative biochemical activities, including nucleotide triphosphatase, nucyleotidylyation and auto-phosphorylation were also investigated in vitro; however Rv1636 could not be shown to definitively possess these activities, raising the possibility that addition factors may be present in vivo.
    [Show full text]
  • Mismatch Repair Inhibits Homeologous Recombination Via Coordinated Directional Unwinding of Trapped DNA Structures
    Molecular Cell Article Mismatch Repair Inhibits Homeologous Recombination via Coordinated Directional Unwinding of Trapped DNA Structures Khek-Chian Tham,1 Nicolaas Hermans,1 Herrie H.K. Winterwerp,3 Michael M. Cox,4 Claire Wyman,1,2 Roland Kanaar,1,2 and Joyce H.G. Lebbink1,2,* 1Department of Genetics, Cancer Genomics Netherlands 2Department of Radiation Oncology Erasmus Medical Center, Rotterdam 3000 CA, The Netherlands 3Division of Biochemistry and Center for Biomedical Genetics, Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands 4Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.07.008 SUMMARY transduction (Zahrt and Maloy, 1997), transformation (Majewski et al., 2000), and conjugational E. coli-E. coli crosses that involve Homeologous recombination between divergent the creation of mismatches (Feinstein and Low, 1986). Likewise, DNA sequences is inhibited by DNA mismatch repair. in eukaryotes, individual MMR proteins have roles of varying In Escherichia coli, MutS and MutL respond to DNA magnitude in the prevention of homeologous recombination mismatches within recombination intermediates (Selva et al., 1995; Datta et al., 1996; Nicholson et al., 2000; de and prevent strand exchange via an unknown mech- Wind et al., 1995). Inhibition of homeologous reactions is thought anism. Here, using purified proteins and DNA sub- to protect the genome from recombination events between divergent repeats (de Wind et al., 1999). This is important in hu- strates, we find that in addition to mismatches within mans, in which the amount of repetitive DNA is as high as 50%. the heteroduplex region, secondary structures within An intriguing question is how MMR and recombination path- the displaced single-stranded DNA formed during ways are integrated at the molecular level.
    [Show full text]
  • Recbcd-Dependent Joint Molecule Formation Promoted by The
    Proc. Nail. Acad. Sci. USA Vol. 88, pp. 3367-3371, April 1991 Biochemistry RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins (single-stranded DNA-binding protein/genetic recombination/homologous pairing) LINDA J. ROMAN, DAN A. DIXON, AND STEPHEN C. KOWALCZYKOWSKI* Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611 Communicated by Bruce M. Alberts, December 18, 1990 (receivedfor review September 22, 1990) ABSTRACT We describe the formation of homologously We previously described a reaction in which heteroduplex paired joint molecules in an in vitro reaction that is dependent DNA formation between ssDNA and homologous linear on the concerted actions ofpurified RecA and RecBCD proteins dsDNA required both the dsDNA-unwinding activity of and is stimulated by single-stranded DNA-binding protein RecBCD and the ssDNA-renaturation activity of RecA (15). (SSB). RecBCD enzyme initiates the process by unwinding the In this paper, we expand upon the previous in vitro studies to linear double-stranded DNA to produce single-stranded DNA, include DNA substrates that are more representative of the which is trapped by SSB and RecA. RecA uses this single- substrates encountered in vivo (i.e., homologous linear ds- stranded DNA to catalyze the invasion of a supercoiled double- DNA and supercoiled dsDNA) and that require the joint stranded DNA molecule, forming a homologously paired joint molecule formation activity of RecA protein. RecA alone is molecule. At low RecBCD enzyme concentrations, the rate- unable to catalyze pairing between two dsDNA molecules limiting step is the unwinding of duplex DNA by RecBCD, unless one of them contains ssDNA in a region of homology whereas at higher RecBCD concentrations, the rate-limiting (16).
    [Show full text]
  • High Fidelity of Reca-Catalyzed Recombination: a Watchdog of Genetic Diversity
    High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity Dror Sagi, Tsvi Tlusty and Joel Stavans Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT delicately balanced, to give the highest chance to the functional maintenance of coded proteins. Furthermore, the Homologous recombination plays a key role in extent of recombination between related organisms, such as generating genetic diversity, while maintaining Escherichia coli and Salmonella, controls their genetic isola- protein functionality. The mechanisms by which tion and speciation (3). Therefore, inhibition of chromosomal RecA enables a single-stranded segment of DNA gene transfer isolates related species. to recognize a homologous tract within a whole In prokaryotes, recombination is carried out by RecA (4), a genome are poorly understood. The scale by which protein that also plays a fundamental role during the repair homology recognition takes place is of a few tens of and bypass of DNA lesions, enabling the resumption of base pairs, after which the quest for homology is DNA replication at stalled replication forks. According to over. To study the mechanism of homology recogni- the prevailing view, the RecA-catalyzed recombination tion, RecA-promoted homologous recombination process proceeds along the following steps. First, a nucleo- protein complex is formed by the polymerization of RecA between short DNA oligomers with different degrees along a single-stranded DNA (ssDNA) substrate. ssDNA, of heterology was studied in vitro, using fluores- which is accepted to be a prerequisite, is produced by the cence resonant energy transfer. RecA can detect recombination-specific helicases RecBCD and RecG at single mismatches at the initial stages of recombi- double strand breaks (5), or as a byproduct of DNA repair nation, and the efficiency of recombination is processes at stalled replication forks (6).
    [Show full text]
  • The Specificity of the Secondary DNA Binding Site of Reca Protein
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10673-10678, October 1996 Biochemistry The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange (genetic recombination/homologous pairing/DNA-protein interactions) ALEXANDER V. MAZIN* AND STEPHEN C. KOWALCZYKOWSKIt Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665 Communicated by Frank Stahl, University of Oregon, Eugene, OR, July 1, 1996 (received for review April 17, 1996) ABSTRACT The RecA protein-single-stranded DNA They suggested that the observed binding specificity is deter- (ssDNA) filament can bind a second DNA molecule. Binding mined by non-Watson-Crick bonds that are formed between of ssDNA to this secondary site shows specificity, in that ssDNA in the primary site and ssDNA bound to the secondary polypyrimidinic DNA binds to the RecA protein-ssDNA fila- site of the nucleoprotein filament, so-called "self-recognition," ment with higher affinity than polypurinic sequences. The as a part of a homology search process. affinity of ssDNA, which is identical in sequence to that bound Despite rather extensive characterization, the precise nature in the primary site, is not always greater than that of and function of the secondary DNA binding site remains nonhomologous DNA. Moreover, this specificity of DNA bind- unknown. Consequently, we sought to determine its role in ing does not depend on the sequence of the DNA bound to the DNA strand exchange and the basis for the apparently novel RecA protein primary site. We conclude that the specificity sequence-specificity attributed to this site.
    [Show full text]
  • Mismatch Repair Proteins Muts and Mutl Inhibit Reca-Catalyzed Strand
    Proc. Natd. Acad. Sci. USA Vol. 91, pp. 3238-3241, April 1994 Biochemistry Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs LEROY WORTH, JR.*, SUSANNA CLARK*, MIROSLAV RADMANt, AND PAUL MODRICH*I *Department of Biochemistry, Duke University Medical Center, Durham, NC 27710; and tLaboratoire de Mutagenese, Institut Jacques Monod, 2 Place Jussieu, Tour 43, 75251 Paris-C6dex 05, France Contributed by Paul Modrich, December 29, 1993 ABSTRACT Bacterial mutS and mutL mutatons confer are 20%o divergent at the sequence level (6), and also controls large increases in recombination between sequences that are the frequency of chromosomal rearrangements occurring in divergent by several percent at the nucleotide level, an effect E. coli as a consequence ofrecombination between rhs repeat attributed to a role for products of these genes In control of elements, which are about 1% divergent at the sequence level recombinatlon fidelity. Since MutS and MutL are proteins (8). Recombination between homeologous sequences scored involved in the eriest steps of mismatch repair, Inclug in either of these systems is increased 1-2 orders of magni- mismatch recognition by MutS, we have tested the psibility tude in mutL or mutS mutants. that they may affect strand exchange in response to occurrence In regular genetic crosses between isogenic genomes, of mspairs within the recombation heteroduplex. We show where the only sources of mismatches are the genetic mark- that MutS aboies RecA-catalyzed strand transfer between fd ers used to score recombinants, one can observe so-called and M13 bacteriophage DNAs, which vary by 3% at the marker effects, which depend on the activity of mismatch- nucleoide level, but Is without effect on M13-M13 or fd-fd repair systems, but appear only when the markers are close exchange.
    [Show full text]
  • Mismatch Repair This Is Backup to Replicative Proofreading By
    Mismatch repair This is backup to replicative proofreading by correcting mismatched nucleotides remaining in DNA after replication. Mismatches often involve the normal four bases in DNA. For example, a T may be mis-paired with a G. Because both T and G are normal components of DNA, mismatch repair systems need some way to determine whether the T or the G is the correct base at a given site. The repair system makes this distinction by identifying the template strand, which contains the original nucleotide sequence, and the newly synthesized strand, which contains the misincorporated base (the error). In bacteria, this distinction can be made based on the pattern of methylation in newly replicated DNA. In E. coli, the A in GATC sequences is methylated subsequent to its synthesis. Thus, a time interval occurs during which the template strand is methylated, and the newly synthesized strand is unmethylated. The mismatch repair system uses this difference in methylation state to excise the mismatched nucleotide in the nascent strand and replace it with the correct nucleotide by using the methylated parental strand of DNA as template. • In E. coli, mismatch repair requires the products of four genes, mutH, mutL, mutS, and mutU (uvrD). • The MutS protein recognizes mismatches and binds to them to initiate the repair process. • MutH and MutL proteins then join the complex. • MutH contains a GATC-specific endonuclease activity that cleaves the unmethylated strand at hemimethylated (that is, half methylated) GATC sites either 5’ or 3’ to the mismatch. The incision sites may be 1000 nucleotide pairs or more from the mismatch.
    [Show full text]
  • Dissecting the Reca-(In)Dependent Response to Mitomycin C in Mycobacterium Tuberculosis Using Transcriptional Profiling and Proteomics Analyses
    cells Article Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses Anna Brzostek 1,*,† , Przemysław Płoci ´nski 1,2,† , Alina Minias 1 , Aneta Ciszewska 1, Filip G ˛asior 1 , Jakub Pawełczyk 1 , Bozena˙ Dziadek 3, Marcin Słomka 4 and Jarosław Dziadek 1,* 1 Institute of Medical Biology of the Polish Academy of Sciences, Lodowa 106, 93-232 Łód´z,Poland; [email protected] (P.P.); [email protected] (A.M.); [email protected] (A.C.); fi[email protected] (F.G.); [email protected] (J.P.) 2 Department of Immunology and Infectious Biology, Faculty of Biology and Environmental Protection, University of Łód´z,Banacha 12/16, 90-237 Łód´z,Poland 3 Department of Molecular Microbiology, Faculty of Biology and Environmental Protection, University of Łód´z, Banacha 12/16, 90-237 Łód´z,Poland; [email protected] 4 Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łód´z,Pomorska 139, 90-235 Łód´z,Poland; [email protected] * Correspondence: [email protected] (A.B.); [email protected] (J.D.) † These authors equally contributed to this work. Abstract: Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, Citation: Brzostek, A.; Płoci´nski,P.; such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of Minias, A.; Ciszewska, A.; G ˛asior, F.; infection while residing inside host macrophages.
    [Show full text]
  • Role of Reca in the Protection of DNA Damage by UV-A in Escherichia Coli
    Journal of Experimental Microbiology and Immunology Vol. 12:39-44 Copyright © April 2008, M&I UBC Role of RecA in the Protection of DNA Damage by UV-A in Escherichia coli. Amanda Chau, Karen Giang, Marcus Leung and Natalie Tam Department of Microbiology and Immunology, UBC Solar Water Disinfection (SODIS) is a simple, yet effective method for water decontamination by utilizing heat and UV-A from sunlight to kill microbes. However, the mechanism for UV-A mediated cell death is unknown. UV-A has been shown to cause DNA damage by the induction of reactive oxygen species (ROS). This damage might cause cell death in SODIS. Therefore, we investigated whether limiting the repair of DNA damaged by UV-A can increase bacterial susceptibility to UV-A. RecA is a DNA repair regulator protein that is activated upon stresses such as heat and reactive oxygen. In this study, we used Escherichia coli strains with and without RecA to determine survival frequencies after UV-A irradiation. recA+ cells with functional RecA showed a higher survival frequency compared to RecA- mutants after 120 minutes of UV-A exposure, while a smaller difference was observed when cells were cultured without light. These results suggest that the absence of RecA is correlated with an increased vulnerability of cells under UV-A exposure. This difference in sensitivity is most likely due to the inability of mutant bacteria to repair DNA damages. Further studies are required to determine not only the effects of RecA on repairing DNA damage, but also other bactericidal mechanisms exerted by UV-A because the presence of RecA was insufficient to fully protect the treated cells.
    [Show full text]