Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA binding modes.


:The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.






Rajendran S,Jezewska MJ,Bujalowski W




Has Abstract


2001-10-02 00:00:00














  • A remote substrate docking mechanism for the tec family tyrosine kinases.

    abstract::During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk ...


    pub_type: 杂志文章


    authors: Joseph RE,Min L,Xu R,Musselman ED,Andreotti AH

    更新日期:2007-05-08 00:00:00

  • Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements.

    abstract::Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNas...


    pub_type: 杂志文章


    authors: Chen SM,Marshall AG

    更新日期:1986-09-09 00:00:00

  • Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering.

    abstract::The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an...


    pub_type: 杂志文章


    authors: Perkins SJ,Nealis AS,Dunham DG,Hardingham TE,Muir IH

    更新日期:1991-11-05 00:00:00

  • A "Seleno Effect" Differentiates the Roles of Redox Active Cysteine Residues in Plasmodium falciparum Thioredoxin Reductase.

    abstract::Here, we introduce the concept of the "seleno effect" in the study of oxidoreductases that catalyze thiol/disulfide exchange reactions. In these reactions, selenium can replace sulfur as a nucleophile, electrophile, or leaving group, and the resulting change in rate (the seleno effect) is defined as kS/ kSe. In soluti...


    pub_type: 杂志文章


    authors: O'Keefe JP,Dustin CM,Barber D,Snider GW,Hondal RJ

    更新日期:2018-03-20 00:00:00

  • NMR solution structure of a synthetic troponin C heterodimeric domain.

    abstract::The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides ...


    pub_type: 杂志文章


    authors: Shaw GS,Sykes BD

    更新日期:1996-06-11 00:00:00

  • Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties.

    abstract::Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of l-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy interve...


    pub_type: 杂志文章


    authors: Tramonti A,Nardella C,di Salvo ML,Barile A,Cutruzzolà F,Contestabile R

    更新日期:2018-12-26 00:00:00

  • Autocatalytically fragmented light chain of botulinum a neurotoxin is enzymatically active.

    abstract::The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc reta...


    pub_type: 杂志文章


    authors: Ahmed SA,McPhie P,Smith LA

    更新日期:2003-11-04 00:00:00

  • Nucleosome Histone Tail Conformation and Dynamics: Impacts of Lysine Acetylation and a Nearby Minor Groove Benzo[a]pyrene-Derived Lesion.

    abstract::Histone tails in nucleosomes play critical roles in regulation of many biological processes, including chromatin compaction, transcription, and DNA repair. Moreover, post-translational modifications, notably lysine acetylation, are crucial to these functions. While the tails have been intensively studied, how the stru...


    pub_type: 杂志文章


    authors: Fu I,Cai Y,Geacintov NE,Zhang Y,Broyde S

    更新日期:2017-04-11 00:00:00

  • Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase.

    abstract::Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during...


    pub_type: 杂志文章


    authors: Xiang J,Jung JY,Sampson NS

    更新日期:2004-09-14 00:00:00

  • Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol.

    abstract::Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is ver...


    pub_type: 杂志文章


    authors: Anandarajah K,Kiefer PM Jr,Donohoe BS,Copley SD

    更新日期:2000-05-09 00:00:00

  • Evidence for an unprecedented histidine hydroxyl modification on D2-His336 in Photosystem II of Thermosynechoccocus vulcanus and Thermosynechoccocus elongatus.

    abstract::The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus vulcanus with a 1.9 Å resolution (PDB: 3ARC ) exhibits, in the two monomers in the asymmetric unit cell, an, until now, unidentified and uninterpreted strong difference in electron density centered at a distance of around 1.5 Å from ...


    pub_type: 杂志文章


    authors: Sugiura M,Koyama K,Umena Y,Kawakami K,Shen JR,Kamiya N,Boussac A

    更新日期:2013-12-31 00:00:00

  • Casein kinase II phosphorylation of caldesmon downregulates myosin-caldesmon interactions.

    abstract::It is well-known that caldesmon (CaD) is a substrate for casein kinase II (CKII), and the phosphorylation of CaD by CKII regulates the interaction of CaD with myosin. However, the functionally relevant CKII phosphorylation site(s) on CaD and the precise role of CaD phosphorylation by CKII in mediating CaD's function h...


    pub_type: 杂志文章


    authors: Wang Z,Yang ZQ

    更新日期:2000-09-12 00:00:00

  • Backbone dynamics of a model membrane protein: 13C NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein.

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    pub_type: 杂志文章


    authors: Henry GD,Weiner JH,Sykes BD

    更新日期:1986-02-11 00:00:00

  • Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 1. Reaction products.

    abstract::The in vitro reaction of the vinyl chloride metabolite chloroacetaldehyde (CAA) with cytosine and adenine residues in ribo- and deoxyribopolynucleotides leads to the formation of the relatively stable hydrated etheno derivatives 3,N4-(N4-alpha-hydroxyethene)adenine (epsilon C . H2O) and 1,N6-(N6-alpha-hydroxyethene)cy...


    pub_type: 杂志文章


    authors: Kuśmierek JT,Singer B

    更新日期:1982-10-26 00:00:00

  • Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling.

    abstract::Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characteriz...


    pub_type: 杂志文章


    authors: Yu C,Gui C,Luo H,Chen L,Zhang L,Yu H,Yang S,Jiang W,Shen J,Shen X,Jiang H

    更新日期:2005-02-08 00:00:00

  • Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit.

    abstract::Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T al...


    pub_type: 杂志文章


    authors: Hingorani VN,Ho Y

    更新日期:1987-03-24 00:00:00

  • Nuclear magnetic resonance solution structure of an undecanucleotide duplex with a complementary thymidine base opposite a 10R adduct derived from trans addition of a deoxyadenosine N6-amino group to (-)-(7R,8S,9R,10S)-7,8-dihydroxy- 9,10-epoxy-7,8,9,10-t

    abstract::The solution structure of a modified undecamer duplex containing (-)-(7R,8S,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene covalently bonded through trans ring opening at C10 of the epoxide by the N6-amino group of deoxyadenosine (dA) was studied. This diol epoxide 1 diastereomer has the benzylic ...


    pub_type: 杂志文章


    authors: Schurter EJ,Sayer JM,Oh-hara T,Yeh HJ,Yagi H,Luxon BA,Jerina DM,Gorenstein DG

    更新日期:1995-07-18 00:00:00

  • Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.

    abstract::Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patien...


    pub_type: 杂志文章,评审


    authors: Cheung JC,Deber CM

    更新日期:2008-02-12 00:00:00

  • Altered K+ channel subunit composition following hormone induction of Kv1.5 gene expression.

    abstract::Diverse voltage-gated K+ channels are produced by tetramerization of channel alpha subunits that can be encoded by multiple genes. In GH3 pituitary cells, the Kv1.4 and Kv1.5 subunit genes are constitutively expressed, and dexamethasone selectively up-regulates expression of the latter gene. Here we use subunit-specif...


    pub_type: 杂志文章


    authors: Takimoto K,Levitan ES

    更新日期:1996-11-12 00:00:00

  • Deep-UV resonance Raman analysis of the Rhodobacter capsulatus cytochrome bc₁complex reveals a potential marker for the transmembrane peptide backbone.

    abstract::Classical strategies for structure analysis of proteins interacting with a lipid phase typically correlate ensemble secondary structure content measurements with changes in the spectroscopic responses of localized aromatic residues or reporter molecules to map regional solvent environments. Deep-UV resonance Raman (DU...


    pub_type: 杂志文章


    authors: Halsey CM,Oshokoya OO,Jiji RD,Cooley JW

    更新日期:2011-08-02 00:00:00

  • Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation.

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    pub_type: 杂志文章


    authors: Grab LA,Swanson BA,Ortiz de Montellano PR

    更新日期:1988-06-28 00:00:00

  • Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site.

    abstract::We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cle...


    pub_type: 杂志文章


    authors: Han J,Burke JM

    更新日期:2005-05-31 00:00:00

  • Incorrect folding of steroidogenic acute regulatory protein (StAR) in congenital lipoid adrenal hyperplasia.

    abstract::Steroidogenic acute regulatory protein (StAR) rapidly stimulates the movement of cholesterol into adrenal and gonadal mitochondria to mediate the acute steroidogenic response; StAR mutations cause potentially lethal congenital lipoid adrenal hyperplasia (lipoid CAH). Bacterially expressed wild-type StAR and four amino...


    pub_type: 杂志文章


    authors: Bose HS,Baldwin MA,Miller WL

    更新日期:1998-07-07 00:00:00

  • Acoustically mounted microcrystals yield high-resolution X-ray structures.

    abstract::We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micromete...


    pub_type: 杂志文章


    authors: Soares AS,Engel MA,Stearns R,Datwani S,Olechno J,Ellson R,Skinner JM,Allaire M,Orville AM

    更新日期:2011-05-31 00:00:00

  • Denaturation of type I collagen fibrils is an endothermic process accompanied by a noticeable change in the partial heat capacity.

    abstract::Thermal transitions of type I collagen fibrils were investigated by differential scanning calorimetry and spectrophotometry of turbidity within a wide range of external conditions. The advanced microcalorimeter allowed us to carry out the measurements at low concentrations of collagen (0.15-0.3 mg/mL). At these concen...


    pub_type: 杂志文章


    authors: Tiktopulo EI,Kajava AV

    更新日期:1998-06-02 00:00:00

  • Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase.

    abstract::The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of C...


    pub_type: 杂志文章


    authors: Geng J,Weitz AC,Dornevil K,Hendrich MP,Liu A

    更新日期:2020-08-04 00:00:00

  • Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors.

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    pub_type: 杂志文章


    authors: Arias HR,Bhumireddy P,Spitzmaul G,Trudell JR,Bouzat C

    更新日期:2006-02-21 00:00:00

  • Following the rotational trajectory of the principal hydrodynamic frame of a protein using multiple probes.

    abstract::A generalized set of fluorescence polarization intensities and electron paramagnetic resonance (EPR) spectra from multiple fluorescent and EPR probes of a specific site on an oriented and immobilized protein element in a biological assembly, combined with anisotropic rotational relaxation studies of the purified and l...


    pub_type: 杂志文章


    authors: Burghardt TP,Ajtai K

    更新日期:1994-05-10 00:00:00

  • Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin.

    abstract::Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degra...


    pub_type: 杂志文章


    authors: Schaefer AV,Leslie BA,Rischke JA,Stafford AR,Fredenburgh JC,Weitz JI

    更新日期:2006-04-04 00:00:00

  • Transformation of the CuA redox site in cytochrome c oxidase into a mononuclear copper center.

    abstract::Subunit II of the aa3 type cytochrome c oxidase contains a binuclear copper center (CuA) which functions as the entry point for electrons donated by cytochrome c. We have introduced site-specific mutations in residues liganding the CuA center in the oxidase of the bacterium Paracoccus denitrificans; the purified, full...


    pub_type: 杂志文章


    authors: Zickermann V,Wittershagen A,Kolbesen BO,Ludwig B

    更新日期:1997-03-18 00:00:00