Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Abstract:

:Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).

journal_name

Biochemistry

journal_title

Biochemistry

authors

Iakoucheva LM,Walker RK,van Houten B,Ackerman EJ

doi

10.1021/bi011041q

subject

Has Abstract

pub_date

2002-01-08 00:00:00

pages

131-43

issue

1

eissn

0006-2960

issn

1520-4995

pii

bi011041q

journal_volume

41

pub_type

杂志文章
  • Equilibrium folding studies of tetrameric R67 dihydrofolate reductase.

    abstract::R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded enzyme that confers resistance to the antibacterial drug trimethoprim. This enzyme is not homologous in sequence or structure to chromosomal DHFRs. Equilibrium folding of tetrameric R67 DHFR was studied and found to be fully reversible. Formation of an inactiv...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00180a018

    authors: Zhuang P,Eisenstein E,Howell EE

    更新日期:1994-04-12 00:00:00

  • Structural consequences of tumor-derived mutations in p16INK4a probed by limited proteolysis.

    abstract::The cyclin-dependent kinase inhibitor p16(INK4a) (hereafter p16) functions as a multiple tumor suppressor. Mutations in p16, which are distributed throughout the entire protein, have been identified in a variety of human cancers and cancer-derived cell lines. It is unclear how tumor-derived mutations disrupt the struc...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi0117100

    authors: Zhang B,Peng ZY

    更新日期:2002-05-21 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...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.8b01074

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

    更新日期:2018-12-26 00:00:00

  • Purification and characterization of an alpha-glucosidase from Saccharomyces carlsbergensis.

    abstract::alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00615a011

    authors: Needleman RB,Federoff HJ,Eccleshall TR,Buchferer B,Marmur J

    更新日期:1978-10-31 00:00:00

  • 9-cis-retinoids: biosynthesis of 9-cis-retinoic acid.

    abstract::Retinoids function through conformational alterations of ligand-dependent nuclear transcription factors, the retinoic acid receptors, and retinoid X receptors. 9-cis-Retinoic acid is a known biological ligand for retinoid X receptors, but its synthesis pathway in vivo is largely unknown. Recently, we identified a cis-...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi992152g

    authors: Paik J,Vogel S,Piantedosi R,Sykes A,Blaner WS,Swisshelm K

    更新日期:2000-07-11 00:00:00

  • Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1.

    abstract::Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS repre...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00455a017

    authors: Picorel R,Lu T,Holt RE,Cotton TM,Seibert M

    更新日期:1990-01-23 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

  • Estrogen-induced changes in high-energy phosphate metabolism in rat uterus: 31P NMR studies.

    abstract::Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found t...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00307a006

    authors: Degani H,Shaer A,Victor TA,Kaye AM

    更新日期:1984-06-05 00:00:00

  • Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

    abstract::The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00590a031

    authors: Viggiano G,Ho NT,Ho C

    更新日期:1979-11-13 00:00:00

  • Renaturation of formiminotransferase-cyclodeaminase from guanidine hydrochloride. Quaternary structure requirements for the activities and polyglutamate specificity.

    abstract::Formiminotransferase-cyclodeaminase denatured in 6 M guanidine hydrochloride (Gdn.HCl) refolds and reassembles to the native octameric structure upon dilution into buffer. Both enzymic activities are recovered to greater than 90%, and the renatured enzyme "channels" the formiminotetrahydropteroylpentaglutamate interme...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00409a042

    authors: Findlay WA,MacKenzie RE

    更新日期:1988-05-03 00:00:00

  • A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.

    abstract::Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. There...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi047717+

    authors: Silva RA,Hilliard GM,Fang J,Macha S,Davidson WS

    更新日期:2005-03-01 00:00:00

  • Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 1. Evidence from deuterium NMR measurements.

    abstract::Deuterium NMR has been used to investigate the structure and dynamic state of cytochrome c complexed with bilayers of cardiolipin. Reductive methylation was employed to prepare [N epsilon, N epsilon-C2H3]lysyl cytochrome c, and deuterium exchange provided labeling of backbone sites to give [amide-2H]cytochrome c or mo...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00230a010

    authors: Spooner PJ,Watts A

    更新日期:1991-04-23 00:00:00

  • Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.

    abstract::We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00416a027

    authors: Tindall KR,Kunkel TA

    更新日期:1988-08-09 00:00:00

  • Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor.

    abstract::Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic p...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00109a020

    authors: Lentz TL

    更新日期:1991-11-12 00:00:00

  • The Q-loop of DrrA is involved in producing the closed conformation of the nucleotide binding domains and in transduction of conformational changes between DrrA and DrrB.

    abstract::DrrA and DrrB proteins form an ATP-dependent efflux pump for doxorubicin and daunorubicin in Streptomyces peucetius. DrrA, the catalytic subunit, forms a complex with the integral membrane protein DrrB. Previous studies have provided evidence for strong interaction between these two proteins, which was found to be cri...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi701699a

    authors: Rao DK,Kaur P

    更新日期:2008-03-04 00:00:00

  • The energy landscape of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

    abstract::3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P, a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria. Earlier studies have established that the condensation occurs with a syn addit...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi901341h

    authors: Tao P,Gatti DL,Schlegel HB

    更新日期:2009-12-15 00:00:00

  • Rate-limiting step: a quantitative definition. Application to steady-state enzymic reactions.

    abstract::The generality of the concept of a rate-limiting step in enzymic reactions recently has been questioned [Northrop, D. B. (1981) Biochemistry 20, 4056-4061] because, in simulated isotopic experiments, alterations of the step identified as rate limiting by current definitions do not consistently affect Vmax in the expec...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00289a003

    authors: Ray WJ Jr

    更新日期:1983-09-27 00:00:00

  • Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum.

    abstract::One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacteriu...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi300409m

    authors: Balashov SP,Petrovskaya LE,Lukashev EP,Imasheva ES,Dioumaev AK,Wang JM,Sychev SV,Dolgikh DA,Rubin AB,Kirpichnikov MP,Lanyi JK

    更新日期:2012-07-24 00:00:00

  • Structural domains of apolipoprotein(a) and its interaction with apolipoprotein B-100 in the lipoprotein(a) particle.

    abstract::The structural domains of human apolipoprotein(a) [apo(a)] and its interaction with apolipoprotein B-100 (apo B-100) in the lipoprotein(a) [Lp(a)] particle were investigated by limited proteolysis with thermolysin and cathepsin D. We characterized the proteolytic products by sodium dodecyl sulfate-polyacrylamide gradi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00177a026

    authors: Huby T,Doucet C,Dieplinger H,Chapman J,Thillet J

    更新日期:1994-03-22 00:00:00

  • Magnetic circular dichroism studies on the mononuclear ferrous active site of phthalate dioxygenase from Pseudomonas cepacia show a change of ligation state on substrate binding.

    abstract::Phthalate dioxygenase from Pseudomonas cepacia contains a mononuclear ferrous center that is strictly required for catalytic oxygen activation. The spectroscopic characterization of this iron site and its ligand interactions has been complicated in the past by interference from a Rieske-type binuclear (2Fe-2S) cluster...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00069a017

    authors: Gassner GT,Ballou DP,Landrum GA,Whittaker JW

    更新日期:1993-05-11 00:00:00

  • Interaction between Cellobiose Dehydrogenase and Lytic Polysaccharide Monooxygenase.

    abstract::Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH de...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.8b01178

    authors: Laurent CVFP,Breslmayr E,Tunega D,Ludwig R,Oostenbrink C

    更新日期:2019-03-05 00:00:00

  • Zn(2+)-dependent misfolding of the p53 DNA binding domain.

    abstract::The DNA binding domain (DBD) of p53 folds by a complex mechanism that involves parallel pathways and multiple intermediates, both on- and off-pathway. This heterogeneity renders DBD particularly susceptible to misfolding and aggregation. The origins of parallel folding mechanisms are not well understood. DBD folding h...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi062106y

    authors: Butler JS,Loh SN

    更新日期:2007-03-13 00:00:00

  • Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations.

    abstract::Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphoryl...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00448a013

    authors: Palczewski K,Arendt A,McDowell JH,Hargrave PA

    更新日期:1989-10-31 00:00:00

  • Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding.

    abstract::IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi020680d

    authors: Arighi CN,Rossi JP,Delfino JM

    更新日期:2003-06-24 00:00:00

  • Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction.

    abstract::Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b00214

    authors: Reynolds KA,Cameron CE,Raney KD

    更新日期:2015-07-14 00:00:00

  • Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

    abstract::The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interf...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00243a037

    authors: Ghomashchi F,Yu BZ,Berg O,Jain MK,Gelb MH

    更新日期:1991-07-23 00:00:00

  • NmerA of Tn501 mercuric ion reductase: structural modulation of the pKa values of the metal binding cysteine thiols.

    abstract::To avoid nonspecific and/or undesirable binding and reactivity of metal ions with cellular components, organisms have evolved metal-specific systems for trafficking proteins. Although systems differ, those handling soft metal ions such as Hg(2+), Cu(+), Zn(2+), etc., all utilize heavy metal-associated (HMA) proteins a...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi100537f

    authors: Ledwidge R,Hong B,Dötsch V,Miller SM

    更新日期:2010-10-19 00:00:00

  • Asymmetric Roles of Two Histidine Residues in Streptococcus pyogenes Cas9 Catalytic Domains upon Chemical Rescue.

    abstract::CRISPR-Cas9 technology has been at the forefront of the field of biology. The Streptococcus pyogenes (SpyCas9) protein forms a complex with guide RNA and can recognize and cleave double-stranded DNA through hybridization based on 20 base pairings. SpyCas9 has two nuclease domains, HNH and RuvC, each of which cuts each...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.0c00766

    authors: Furuhata Y,Kato Y

    更新日期:2021-01-26 00:00:00

  • Proton magnetic resonance studies of carbonic anhydrase. III. Binding of sulfonamides.

    abstract::Resonances of the histidine region of human carbonic anhydrase B have been studied by proton magnetic resonance spectroscopy in the presence of seven sulfonamide inhibitors. Results of difference spectroscopy and observation of the C-2 resonance of an additional titratable histidine in some of these spectra suggest a ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00675a007

    authors: Pesando JM,Grollman AP

    更新日期:1975-02-25 00:00:00

  • The interface between the EGF2 domain and the protease domain in blood coagulation factor IX contributes to factor VIII binding and factor X activation.

    abstract::The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the proteas...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi060451h

    authors: Fribourg C,Meijer AB,Mertens K

    更新日期:2006-09-05 00:00:00