Abstract:
:After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Bakhtina M,Roettger MP,Kumar S,Tsai MDdoi
10.1021/bi700084wsubject
Has Abstractpub_date
2007-05-08 00:00:00pages
5463-72issue
18eissn
0006-2960issn
1520-4995journal_volume
46pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::The laminin alpha3 chain is mainly expressed at the skin, and its C-terminal G domain has a critical role in multiple biological functions. We screened for biologically active sites on the mouse laminin alpha3 chain G domain using 107 synthetic peptides on coated plates and conjugated to Sepharose beads with HT1080 hu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi901421t
更新日期:2009-11-10 00:00:00
abstract::The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi010525j
更新日期:2001-09-11 00:00:00
abstract::Nitrogenase catalyzes the reduction of N2 to NH3, supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin (in vivo) and sodium dithionite (in vitro). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitroge...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00844
更新日期:2019-11-19 00:00:00
abstract::The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. T...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi8014828
更新日期:2008-11-11 00:00:00
abstract::The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035344r
更新日期:2003-11-25 00:00:00
abstract::The "stalk" of the prokaryotic 50S ribosomal subunit is comprised of four copies of the protein L7/L12. In Escherichia coli, L7/L12 is a dimeric protein at micromolar concentrations, which is able to undergo rapid subunit exchange. A recent structural study indicated a tetrameric arrangement of the L12 proteins isolat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048015n
更新日期:2005-03-08 00:00:00
abstract::Histidinol-phosphate aminotransferase (HspAT) is a key enzyme on the histidine biosynthetic pathway. HspAT catalyzes the transfer of the amino group of L-histidinol phosphate (Hsp) to 2-oxoglutarate to form imidazole acetol phosphate (IAP) and glutamate. Thus, HspAT recognizes two kinds of substrates, Hsp and glutamat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi002769u
更新日期:2001-04-17 00:00:00
abstract::In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi970977p
更新日期:1997-12-02 00:00:00
abstract::Apolipoprotein E is immunochemically localized to amyloid plaque in Alzheimer's brains, and the allelic distribution of ApoE in individuals is associated with a disposition toward Alzheimer's disease. We show here that all three ApoE isotypes exhibit a strong and specific ability to inhibit both nucleation and seeding...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961074j
更新日期:1996-09-24 00:00:00
abstract::Apolipophorin III (apoLp-III) from the migratory locust, Locusta migratoria, represents the only full-length apolipoprotein whose three-dimensional structure has been solved. In the present study, spectroscopic methods have been employed to investigate the effects of deglycosylation (via endoglycosidase F treatment) a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00178a019
更新日期:1994-03-29 00:00:00
abstract::The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001549d
更新日期:2001-02-06 00:00:00
abstract::The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donati...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00161a024
更新日期:1992-11-24 00:00:00
abstract::Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting p...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061837v
更新日期:2006-12-26 00:00:00
abstract::To identify interactions between Ste2p, a G protein-coupled receptor of the yeast Saccharomyces cerevisiae, and its tridecapeptide ligand, alpha-factor (WHWLQLKPGQPMY), a variety of alpha-factor analogues were used in conjunction with site-directed mutagenesis of a targeted portion of Ste2p transmembrane domain six. A...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi026100u
更新日期:2002-11-19 00:00:00
abstract::The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vib...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00492a012
更新日期:1990-10-09 00:00:00
abstract::In this study, we prepared several shortened and full-length insulin analogues with substitutions at position B26. We compared the binding affinities of the analogues for rat adipose membranes with their ability to lower the plasma glucose level in nondiabetic Wistar rats in vivo after subcutaneous administration, and...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi702086w
更新日期:2008-05-27 00:00:00
abstract::Recently, we described the formation of a complex between liposomes and the unfolded protein rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), which could be liberated and efficiently reactivated after treatment of the complex with detergents [Zardeneta, G., & Horowitz, P. M. (1992) Eur. J. Biochem. 210, ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00213a025
更新日期:1993-12-21 00:00:00
abstract::Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins. We recently reported the identification of the gene cluster responsible for the biosynthesis of these toxins in cyanobacteria and proposed a biosynthetic scheme starting from L-proline and involving three polyketide synthases for which the starte...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9018785
更新日期:2010-01-12 00:00:00
abstract::We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi980097q
更新日期:1998-05-26 00:00:00
abstract::There are a small number of peptides derived from proteins that have a propensity to adopt structure in aqueous solution which is similar to the structure they possess in the parent protein. There are far fewer examples of protein fragments which adopt stable nonnative structures in isolation. Understanding how nonnat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi990320z
更新日期:1999-06-01 00:00:00
abstract::A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxid...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi010615o
更新日期:2001-10-16 00:00:00
abstract::Cells can be transiently permeabilized by application of high electric pulses of short duration. A direct consequence of this treatment is to induce a fusogenic state in the pulsed membrane. The molecular events underlying these phenomena remain to be explained. During our work, we investigated the effects of pulsing ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00471a009
更新日期:1990-05-15 00:00:00
abstract::We have recently reported that the Na pump alpha-subunit has cytosolic-oriented oligosaccharides which were sensitive to cleavage by an enzyme specific for hydrolysis of N-linked glycans [Pedemonte et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9789-9793]. We now describe experiments that characterize the saccharides...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00158a008
更新日期:1992-11-03 00:00:00
abstract::Tryptic digestion studies of the human erythrocyte glucose carrier have shown that a reactive and transport-sensitive exofacial sulfhydryl is located in the carboxy-terminal half of the molecule, corresponding to Cys347, Cys421, or Cys429. In the present studies, the erythrocyte glucose carrier labeled on the exofacia...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00497a014
更新日期:1990-11-13 00:00:00
abstract::The calcitonin/CGRP gene pre-mRNA is alternatively spliced in a tissue-specific manner resulting in the formation of calcitonin mRNA in thyroid C-cells and CGRP mRNA in neurons. Computer analysis of the RNA containing the 3' splice acceptor of the calcitonin-specific exon 4 predicts that this region has the potential ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9808058
更新日期:1998-11-10 00:00:00
abstract::To understand the mechanism by which annexin I induces membrane aggregation, a comprehensive mutagenesis of all six Ca2+-binding sites was performed. When the cap residues of type II Ca2+-binding sites were systematically mutated to Ala, a type II site in domain II was shown to be essential for Ca2+-dependent vesicle ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi980479w
更新日期:1998-07-14 00:00:00
abstract::High-affinity mu-conotoxin block of skeletal muscle Na+ channels depends on an arginine at position 13 (Arg-13). To understand both the mechanism of toxin interaction and the general structure of its binding site in the channel mouth, we examined by thermodynamic mutant cycle analysis the interaction between the criti...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9724927
更新日期:1998-03-31 00:00:00
abstract::Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048142j
更新日期:2005-02-08 00:00:00
abstract::Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4'-phosphopantetheine (4'-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4'-PP is mediated by 4' phosphopantetheinyl transfersases (PPTases). Mycobacter...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi501444e
更新日期:2015-04-14 00:00:00
abstract::The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured as a function of temperature in dark-adapted photosystem II (PS II) membrane fragments that were reconstituted with native plastoquinone-9 (PQ-9) by a recently developed procedure [Kurrec...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi962302c
更新日期:1997-03-04 00:00:00