Mechanism of translocation and kinetics of DNA unwinding by the helicase RecG.

Abstract:

:RecG is a DNA helicase involved in the repair of damage at a replication fork and catalyzes the reversal of the fork to a point beyond the damage in the template strand. It unwinds duplex DNA in reactions that are coupled to ATP hydrolysis. The kinetic mechanism of duplex DNA unwinding by RecG was analyzed using a quantitative fluorescence assay based on the process of contact quenching between Cy3 and Dabcyl groups attached to synthetic three-way DNA junctions. The data show that the protein moves at a rate of 26 bp s(-1) along the duplex DNA during the unwinding process. RecG ATPase activity during translocation indicates a constant rate of 7.6 s(-1), measured using a fluorescent phosphate sensor, MDCC-PBP. These two rates imply a movement of approximately 3 bp per ATP hydrolyzed. We demonstrate in several trapping experiments that RecG remains attached to DNA after translocation to the end of the arm of the synthetic DNA junction. ATPase activity continues after translocation is complete. Dissociation of RecG from the product DNA occurs only very slowly, suggesting strong interactions between them. The data support the idea that interactions of the duplex template arm with the protein are the major sites of binding and production of translocation.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Martinez-Senac MM,Webb MR

doi

10.1021/bi0512851

subject

Has Abstract

pub_date

2005-12-27 00:00:00

pages

16967-76

issue

51

eissn

0006-2960

issn

1520-4995

journal_volume

44

pub_type

杂志文章
  • Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s.

    abstract::The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum o...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00331a025

    authors: Lennick M,Brew SA,Ingham KC

    更新日期:1985-05-07 00:00:00

  • A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

    abstract::Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi501198f

    authors: Wu EY,Walsh AR,Materne EC,Hiltner EP,Zielinski B,Miller BR 3rd,Mawby L,Modeste E,Parish CA,Barnes WM,Kermekchiev MB

    更新日期:2015-01-27 00:00:00

  • Structural uncoupling between opposing domains of oxidized calmodulin underlies the enhanced binding affinity and inhibition of the plasma membrane Ca-ATPase.

    abstract::Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidat...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi0474113

    authors: Chen B,Mayer MU,Squier TC

    更新日期:2005-03-29 00:00:00

  • Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding.

    abstract::The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate p...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00141a004

    authors: Ullner M,Selander M,Persson E,Stenflo J,Drakenberg T,Teleman O

    更新日期:1992-07-07 00:00:00

  • Activation of epidermal growth factor receptor by epidermal growth factor.

    abstract::The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) induces dimerization of the receptor and activation of its protein tyrosine kinase. Each of these three steps was followed as a function of the concentrations of EGF and of EGF receptor. Binding of EGF was followed by sedim...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9602268

    authors: Sherrill JM,Kyte J

    更新日期:1996-05-07 00:00:00

  • Thermal unfolding of triosephosphate isomerase from Entamoeba histolytica: dimer dissociation leads to extensive unfolding.

    abstract::In mesophiles, triosephosphate isomerase (TIM) is an obligated homodimer. We have previously shown that monomeric folding intermediates are common in the chemical unfolding of TIM, where dissociation provides 75% of the overall conformational stability of the dimer. However, analysis of the crystallographic structure ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi801360k

    authors: Tellez LA,Blancas-Mejia LM,Carrillo-Nava E,Mendoza-Hernández G,Cisneros DA,Fernández-Velasco DA

    更新日期:2008-11-04 00:00:00

  • High-resolution proton nuclear magnetic resonance analysis of conformational properties of biosynthetic actinomycin analogues.

    abstract::High-resolution proton nuclear magnetic resonance (1H NMR) has been used to study the conformation and dynamics of biosynthetic analogues of actinomycin D that have been substituted at one or both of the 3'-amino acids with azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline for one or both of the prolines. ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00540a007

    authors: Mirau PA,Shafer RH

    更新日期:1982-05-25 00:00:00

  • Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition.

    abstract::The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00236a025

    authors: Hua QX,Weiss MA

    更新日期:1991-06-04 00:00:00

  • Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum.

    abstract::Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two ind...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi300261p

    authors: Hlouchova K,Rudolph J,Pietari JM,Behlen LS,Copley SD

    更新日期:2012-05-08 00:00:00

  • Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity.

    abstract::Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without s...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi701378g

    authors: Ahn K,Johnson DS,Fitzgerald LR,Liimatta M,Arendse A,Stevenson T,Lund ET,Nugent RA,Nomanbhoy TK,Alexander JP,Cravatt BF

    更新日期:2007-11-13 00:00:00

  • Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.

    abstract::Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00069a009

    authors: Chu E,Takimoto CH,Voeller D,Grem JL,Allegra CJ

    更新日期:1993-05-11 00:00:00

  • Solution-state structure of the Dewar pyrimidinone photoproduct of thymidylyl-(3'----5')-thymidine.

    abstract::The preparation, spectroscopic investigation, structure determination, conformational analysis, and modeling of the Dewar pyrimidinone photoproduct of thymidylyl-(3'----5')-thymidine, previously referred to as TpT3 [Johns, H. E., Pearson, M. L., LeBlanc, J. C., & Heilleiner, C. W. (1964) J. Mol. Biol. 9, 503-524], is ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00419a007

    authors: Taylor JS,Garrett DS,Cohrs MP

    更新日期:1988-09-20 00:00:00

  • Characterization of the properties of ethenoadenosine nucleotides bound or trapped at the active site of myosin subfragment 1.

    abstract::The fluorescent nucleotide analogue of ADP, 1,N6-ethenoadenosine diphosphate (epsilon ADP), has been used to probe the active site of myosin subfragment 1 (SF1). The Mg complex of ADP was shown to be trapped stoichiometrically at the active site by a variety of thiol cross-linking agents having sulfur to sulfur spanni...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00312a029

    authors: Perkins WJ,Wells JA,Yount RG

    更新日期:1984-08-14 00:00:00

  • Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies.

    abstract::Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi0497244

    authors: Wang H,Vath GM,Gleason KJ,Hanna PE,Wagner CR

    更新日期:2004-06-29 00:00:00

  • Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

    abstract::Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The fi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00329a015

    authors: Morero RD,Viñals AL,Bloj B,Farías RN

    更新日期:1985-04-09 00:00:00

  • Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies.

    abstract::The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM et...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00340a040

    authors: Ingraham RH,Swenson CA

    更新日期:1985-09-10 00:00:00

  • Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis.

    abstract::Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formati...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi049821x

    authors: Castro C,Gratson AA,Evans JC,Jiracek J,Collinsová M,Ludwig ML,Garrow TA

    更新日期:2004-05-11 00:00:00

  • Production and characterization of a functional putidaredoxin reductase-putidaredoxin covalent complex.

    abstract::In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi901872s

    authors: Churbanova IY,Poulos TL,Sevrioukova IF

    更新日期:2010-01-12 00:00:00

  • Acetylation of lysine 92 improves the chaperone and anti-apoptotic activities of human αB-crystallin.

    abstract::αB-Crystallin is a chaperone and an anti-apoptotic protein that is strongly expressed in many tissues, including the lens, retina, heart, and kidney. In the human lens, several lysine residues in αB-crystallin are acetylated. We have previously shown that such acetylation is predominant at lysine 92 (K92) and lysine 1...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi400638s

    authors: Nahomi RB,Huang R,Nandi SK,Wang B,Padmanabha S,Santhoshkumar P,Filipek S,Biswas A,Nagaraj RH

    更新日期:2013-11-12 00:00:00

  • Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes.

    abstract::The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi982127v

    authors: Misono KS,Sivasubramanian N,Berkner K,Zhang X

    更新日期:1999-01-12 00:00:00

  • Replication of antibiotic resistance plasmid R6K DNA in vitro.

    abstract::A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and strepto...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00606a017

    authors: Inuzuka M,Helinski DR

    更新日期:1978-06-27 00:00:00

  • Pre-steady-state kinetics reveal a slow isomerization of the enzyme-NAD complex in the NAD-malic enzyme reaction.

    abstract::Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00059a007

    authors: Rajapaksa R,Abu-Soud H,Raushel FM,Harris BG,Cook PF

    更新日期:1993-03-02 00:00:00

  • Photochemical reactions of photosystem II in ethylene glycol.

    abstract::The behavior of photosystem II (PSII) reactions was investigated under conditions of decreasing water content by the addition of increasing concentrations of ethylene glycol (EG). The photosynthetic activities were measured for PSII samples either directly in aqueous solutions of EG or in the standard buffer medium fo...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi961724j

    authors: Hillier W,Lukins P,Seibert M,Wydrzynski T

    更新日期:1997-01-07 00:00:00

  • Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues.

    abstract::The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dep...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00270a013

    authors: Wagner T,Sprinzl M

    更新日期:1983-01-04 00:00:00

  • Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion.

    abstract::Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or bind...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00243a033

    authors: Di Giambattista M,Engelborghs Y,Nyssen E,Clays K,Cocito C

    更新日期:1991-07-23 00:00:00

  • Polyphosphoprotein from the adhesive pads of Mytilus edulis.

    abstract::Achieving a satisfactory biochemical explanation for the opportunistic underwater adhesion of marine invertebrates such as mussels and barnacles requires a detailed characterization of proteins extracted from holdfast structures produced by these organisms. Mefp-5 is an adhesive protein derived from the foot of the co...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi002718x

    authors: Waite JH,Qin X

    更新日期:2001-03-06 00:00:00

  • The high molecular weight nerve growth factor complex from Mastomys natalensis differs from the murine nerve growth factor complex.

    abstract::Both the male and the female of Mastomys natalensis, an African rat, have high levels of nerve growth factor (NGF) in their submaxillary glands. Mastomys high molecular weight (HMW) NGF was purified by gel filtration and ion-exchange chromatography and was compared with HMW NGF from the male mouse submaxillary gland. ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00418a007

    authors: Darling TL,Fahnestock M

    更新日期:1988-09-06 00:00:00

  • Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase.

    abstract::Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic resi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi2012228

    authors: Meints CE,Gustafsson FS,Scrutton NS,Wolthers KR

    更新日期:2011-12-27 00:00:00

  • Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines.

    abstract::Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melt...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00395a006

    authors: Kim JT,Mattai J,Shipley GG

    更新日期:1987-10-20 00:00:00

  • Presence of two rhodopsin intermediates responsible for transducin activation.

    abstract::To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral dat...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi970932o

    authors: Tachibanaki S,Imai H,Mizukami T,Okada T,Imamoto Y,Matsuda T,Fukada Y,Terakita A,Shichida Y

    更新日期:1997-11-18 00:00:00