Abstract:
:We describe a fluorescence assay that can be used to monitor helicase-catalyzed unwinding of duplex DNA continuously in real time. The assay is based on the observation that fluorescence resonance energy transfer (FRET) occurs between donor (fluorescein) and acceptor (hexachlorofluorescein) fluorophores that are in close proximity due to their covalent attachment to the 3' and 5' ends of the complementary strands of a duplex oligodeoxynucleotide. FRET results in a reduction in the fluorescence emission intensity of fluorescein in the duplex DNA substrate relative to that observed for fluorescein-labeled single stranded DNA. Therefore, an enhancement of fluorescein fluorescence (lambda ex = 492 nm; lambda em = 520 nm) occurs upon helicase-catalyzed unwinding of the duplex DNA and separation of the complementary strands. The fluorescence assay is extremely sensitive, allowing DNA unwinding reactions to be monitored continuously at DNA concentrations as low as 1 nM in a fluorescence stopped-flow experiment. We demonstrate the use of this DNA substrate in pre-steady state, single turnover studies of duplex DNA unwinding catalyzed by the Escherichia coli Rep helicase, monitored by fluorescence stopped flow. We show that the fluorescence enhancement monitors Rep-catalyzed DNA unwinding by comparisons with identical kinetic studies carried out using rapid chemical quench-flow techniques. Single turnover kinetic studies performed at 1 nM DNA as a function of excess Rep concentration show that Rep-catalyzed unwinding of an 18 base pair duplex containing a 3'-ss-(dT)20 tail is biphasic and can be described by the sum of two exponential terms. The observed rate constant of the first phase is independent of [Rep] (20-300 nM) and measures the rapid single turnover, unwinding of the duplex DNA by Rep dimers bound in productive complexes (1.3 +/- 0.2 s-1; 23 +/- 3 base pairs s-1 at 25.0 degrees C). The observed rate constant for the second phase increases linearly with [Rep], reflecting DNA unwinding that is limited by a Rep binding event occurring with a bimolecular rate constant of (1.8 +/- 0.1) x 10(5) M-1 s-1, which may reflect the rate constant for Rep dimerization on DNA. Kinetic competition studies indicate that both Rep subunits are bound stably to the DNA substrate in the productive complex that is unwound in the fast phase. The results of these kinetic studies are consistent with an active, rolling mechanism for Rep-catalyzed unwinding of DNA [Wong, I., & Lohman, T. M., (1992) Science 256, 350].(ABSTRACT TRUNCATED AT 400 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Bjornson KP,Amaratunga M,Moore KJ,Lohman TMdoi
10.1021/bi00251a044subject
Has Abstractpub_date
1994-11-29 00:00:00pages
14306-16issue
47eissn
0006-2960issn
1520-4995journal_volume
33pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi981137t
更新日期:1998-10-06 00:00:00
abstract::The cardiac-specific Nkx2.5 homeodomain has been expressed as a 79-residue protein with the oxidizable Cys(56) replaced with Ser. The Nkx2.5 or Nkx2.5(C56S) homeodomain is 73% identical in sequence to and has the same NMR structure as the vnd (ventral nervous system defective)/NK-2 homeodomain of Drosophila when bound...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi050835s
更新日期:2005-09-20 00:00:00
abstract::The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025952r
更新日期:2002-10-29 00:00:00
abstract::The solution conformation of an agalactosyl penta-antennary "bisected" N-linked glycan from hen ovomucoid has been determined using a combination of 1H-NMR NOE measurements and restrained molecular dynamics (MD) simulations. The majority of glycosidic linkages exhibited restricted torsional fluctuations about the glob...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00043a018
更新日期:1995-10-31 00:00:00
abstract::Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dim...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0616706
更新日期:2006-11-28 00:00:00
abstract::Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues w...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi050870z
更新日期:2006-01-24 00:00:00
abstract::Glycerol stabilizes the triple-helical structure of solubilized calf skin collagen. The equilibrium melting temperature of the protein increased linearly from 38.0 degrees C in AS buffer (0.01 M NaOAc and 0.02 M NaCl, pH 4.0) to 43.0 degrees C in AS and 6 M glycerol buffer. To understand the thermodynamic basis of thi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00353a004
更新日期:1986-03-11 00:00:00
abstract::In contrast to all other known pyrophosphatases, Moorella thermoacetica pyrophosphatase (mtCBS-PPase) contains nucleotide-binding CBS domains and is thus strongly regulated by adenine nucleotides. Stopped-flow measurements using a fluorescent AMP analogue, 2'(3')-O-(N-methylanthranoyl)-AMP (Mant-AMP), reveal that nucl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9019737
更新日期:2010-02-09 00:00:00
abstract::The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] clu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi201003a
更新日期:2011-11-01 00:00:00
abstract::alpha5beta1 integrin can occupy several distinct conformational states which support different strengths of binding to fibronectin [García, A. J., et al. (1998) J. Biol. Chem. 273, 34710-34715]. Using a model system in which specific activating monoclonal antibodies were used to achieve uniform activated states, the b...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025752f
更新日期:2002-07-23 00:00:00
abstract::The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschy...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi201500c
更新日期:2012-02-07 00:00:00
abstract::Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatogra...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00396a019
更新日期:1987-11-03 00:00:00
abstract::When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00016a006
更新日期:1995-04-25 00:00:00
abstract::Structural properties of heparan sulfate preparations from hog mucosa and beef lung sources were obtained by application of Smith degradation and nitrous acid reactions. Products formed by these reactions indicated that most of the iduronic acid present in these mucopolysaccharides is ester sulfated, whereas N-sulfate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00629a014
更新日期:1977-05-17 00:00:00
abstract::The interaction of aminooxy compounds such as aminooxyacetate (AAA), L-canaline, and hydroxylamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) was studied by absorption spectra and stopped-flow spectrophotometry and compared with the unique feature of interaction of O-amino-D-serine (OADS) with the e...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00451a010
更新日期:1989-12-12 00:00:00
abstract::The relationship between triacylglycerol and monoacylglycerol hydrolyzing activities of purified rat heart lipoprotein lipase was studied using emulsified trioleoylglycerol and micellar or albumin-bound monooleoylglycerol as substrates. The maximal reaction rates obtained with the two substrates were similar (650 and ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00654a017
更新日期:1976-05-04 00:00:00
abstract::RNA:pseudouridine synthetase (Pus1) from Saccharomyces cerevisiae is a multisite specific enzyme that catalyzes the formation of pseudouridine at positions 34 and 36 of intron-containing precursor tRNAIle and at positions 27 and/or 28 of several yeast tRNAs. In this paper we demonstrate that the purified recombinant P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972671o
更新日期:1998-05-19 00:00:00
abstract::Treatment of human lymphoid cells with methyl methanesulfonate (MMS) results in discontinuities in cellular DNA. Discontinuities can be detected by sedimentation through neutral sucrose gradients after digestion with S1 nuclease on top of the gradient and by benzoylated naphthoylated diethylaminoethylcellulose (BND-ce...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00639a024
更新日期:1977-10-04 00:00:00
abstract::How various anti-amyloidogenic compounds inhibit the formation of Alzheimer's beta-amyloid fibrils (fAbeta) from amyloid beta-peptide (Abeta) and destabilize fAbeta remains poorly understood. Using spectrophotometry, spectrofluorometry, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061540x
更新日期:2007-02-20 00:00:00
abstract::Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylp...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi010410f
更新日期:2001-06-26 00:00:00
abstract::Complex I, a key component of the mitochondrial respiratory chain, exhibits diminished activity as a result of cardiac ischemia/reperfusion. Cardiac ischemia/reperfusion is associated with increases in the levels of mitochondrial Ca(2+) and pro-oxidants. In the current in vitro study, we sought evidence for a mechanis...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049803f
更新日期:2004-07-06 00:00:00
abstract::The side-chain dynamics of methyl groups in two structurally related proteins from the fibronectin type III (fnIII) superfamily, the third fnIII domain from human tenascin (TNfn3) and the tenth fnIII domain from human fibronectin (FNfn10), have been studied by NMR spectroscopy. Side-chain order parameters reveal that ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035658e
更新日期:2004-02-10 00:00:00
abstract::Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971368k
更新日期:1997-10-21 00:00:00
abstract::Calcium protects a limited number of embryonic chick neural retina cell surface proteins from tryptic cleavage. One glycoprotein of Mr approximately 1.3 X 10(5) and pI approximately 4.8 (gp130) is present in intact retinas and, in the presence of Ca2+, is both resistant to tryptic cleavage and poorly iodinated. When c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00300a016
更新日期:1984-02-28 00:00:00
abstract::Xylose isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this m...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049812o
更新日期:2004-06-01 00:00:00
abstract::It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational tr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00118a014
更新日期:1992-01-28 00:00:00
abstract::PI-SceI is a member of an unusual class of rare cutting homing endonucleases produced by an autocatalytic protein splicing from a precursor. To analyze the structural and functional domain organization of the endonuclease PI-SceI and to examine whether the DNA binding activity can be structurally separated from the ca...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi980013d
更新日期:1998-06-02 00:00:00
abstract::The biosynthesis of the antitumor antibiotic sibiromycin by Streptosporangium sibiricum requires the construction of four units: the amino sugar from glucose; the anthranilate ring from DL-tryptophan probably via kynurenine; the aromatic methyl group from methionine; the propylidene proline from L-tyrosine with the lo...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00586a029
更新日期:1979-09-18 00:00:00
abstract::Malondialdehyde (MDA) and nucleobase propenals can transfer oxopropenyl groups to guanine residues of DNA to yield pyrimodopurinone (M(1)G) adducts. The DNA structural requirements for reaction with alpha,beta-unsaturated aldehydes were explored. We found that single-stranded DNA is more sensitive to oxopropenylation ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0113059
更新日期:2002-04-16 00:00:00
abstract::Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of repre...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002297j
更新日期:2001-03-13 00:00:00