Abstract：
:Although Mg(2+) is the cation that functions as the cofactor for the nucleotidyl transfer reaction for almost all DNA polymerases, Mn(2+) can also serve, but when it does, the degree of base discrimination exhibited by most DNA polymerases (pols) is diminished. Metal ions other than Mg(2+) or Mn(2+) can also act as cofactors depending on the specific DNA polymerase. Here, we tested the ability of several divalent metal ions to substitute for Mg(2+) or Mn(2+) with RB69 DNA polymerase (RB69pol), a model B-family pol. Our choice of metal ions was based on previous studies with other DNA pols. Co(2+), and to a lesser extent Ni(2+), were the only cations among those tested besides Mg(2+) and Mn(2+) that could serve as cofactors with RB69pol. The incorporation efficiency of correct dNMPs increased by 5-fold with Co(2+), relative to that of Mg(2+). The incorporation efficiencies of incorrect dNMPs increased by 2-17-fold with Co(2+), relative to that with Mg(2+) depending on the incoming dNTP. Base selectivity was reduced even further with Mn(2+) compared to that observed with Co(2+). Substitution of Mn(2+), Co(2+), or Ni(2+) for Mg(2+) reduced the exonuclease activity of RB69pol by 2-, 6-, and 33-fold, respectively, contributing to the frequency of misincorporation. In addition, Co(2+) and Mn(2+) were better able to extend a primer past a mismatch than Mg(2+). Finally, Co(2+) and Mn(2+) enhanced ground-state binding of both correct and incorrect dNTPs to RB69pol:dideoxy-terminated primer-template complexes.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Vashishtha AK,Konigsberg WHdoi
10.1021/acs.biochem.5b01350subject
Has Abstractpub_date
2016-05-10 00:00:00pages
2661-70issue
18eissn
0006-2960issn
1520-4995journal_volume
55pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract：:A full-length cDNA for dopamine beta-monooxygenase (D beta M) from bovine adrenal glands has been cloned and sequenced. The soluble and membrane-derived forms of D beta M have also been sequenced from their N-termini. While the observed sequences for the soluble protein correspond to those previously reported [Joh, T....
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00452a026
更新日期：1989-12-26 00:00:00
abstract：:TCDD (dioxin) induces a rapid inflammatory response from 3T3-L1 adipocytes as judged by prominent induction of the mRNA expression of prostaglandin-endperoxide synthase 2 (Cox-2) along with other inflammation markers within 1 h. This action of TCDD is clearly antagonized by cell pretreatment with AACOCF3 (an inhibitor...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi801913w
更新日期：2008-12-30 00:00:00
abstract：:Time-resolved resonance Raman (TR(3)) and time-resolved step-scan (TRS(2)) FTIR spectroscopies have been used to probe the structural dynamics at the heme b(3) proximal and distal sites after carbon monoxide photolysis from fully reduced CO-bound nitric oxide reductase. The Raman spectra of the transient species exhib...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi035289m
更新日期：2003-12-23 00:00:00
abstract：:Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi035890k
更新日期：2004-03-16 00:00:00
abstract：:Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in sm...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00547a019
更新日期：1980-03-18 00:00:00
abstract：:A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00356a045
更新日期：1986-04-22 00:00:00
abstract：:The membrane-bound quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli contains pyrroloquinoline quinone (PQQ) and participates in the direct oxidation of D-glucose to D-gluconate by transferring electrons to ubiquinone (UQ). To elucidate the mechanism of ubiquinone reduction by mGDH, we applied a pulse radi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi051347n
更新日期：2005-10-18 00:00:00
abstract：:Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hyd...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00098a007
更新日期：1991-08-27 00:00:00
abstract：:The binding of ferric ions, chelated with nitrilotriacetate, to human serum transferrin (hTF) has been studied using ultrasensitive titration calorimetry. Studies were done in both the presence and the absence of the synergistic bicarbonate anion. It was found that the C-site of hTF is capable of weakly binding bicarb...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00087a019
更新日期：1993-09-14 00:00:00
abstract：:Structural changes in the complex formation between transducin and metarhodopsin II, the activated form of photolyzed rhodopsin, in visual transduction processes were analyzed by Fourier transform infrared spectroscopy. The spectrum of the complex was obtained by subtracting the contribution of metarhodopsin I and unc...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi960911e
更新日期：1996-10-15 00:00:00
abstract：:NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread d...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/acs.biochem.8b00532
更新日期：2018-06-26 00:00:00
abstract：:The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predomina...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00490a029
更新日期：1990-09-25 00:00:00
abstract：:Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Gl...
journal_title：Biochemistry
pub_type： 杂志文章
doi：
更新日期：2001-08-28 00:00:00
abstract：:EL222 is a blue light sensor protein, which consists of a light-oxygen-voltage domain as a light sensor and a LuxR-type helix-turn-helix DNA-binding domain. The reaction dynamics of the protein-DNA binding were observed for the first time using the time-resolved transient grating method. The reaction scheme was determ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/acs.biochem.7b01206
更新日期：2018-03-13 00:00:00
abstract：:High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affec...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00323a016
更新日期：1985-01-15 00:00:00
abstract：:In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopher...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi701419d
更新日期：2007-12-11 00:00:00
abstract：:The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH -->...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi0003691
更新日期：2000-11-28 00:00:00
abstract：:The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thal...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi700426q
更新日期：2007-08-21 00:00:00
abstract：:Guanidinium chloride- (GdmCl-) induced unfolding of beta-lactamase has been investigated by a combination of size-exclusion chromatography (SEC-FPLC) and usual optical methods. It has been shown that at low temperatures this protein unfolds through two equilibrium intermediates. The first of these intermediates is the...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00176a006
更新日期：1994-03-15 00:00:00
abstract：:We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecul...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi051752e
更新日期：2006-02-21 00:00:00
abstract：:The initial hydrolysis of CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rat constant for the first-order process is independe...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00382a035
更新日期：1987-04-21 00:00:00
abstract：:[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resona...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00450a035
更新日期：1989-11-28 00:00:00
abstract：:Induced fit has been postulated to be an important component of ligand interactions with proteins, including protein-DNA interactions. We imagined that the entropic cost of induced fit might be highly dependent on the local protein sequence context around critical contact residues. To investigate this question, we ana...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi960256a
更新日期：1996-06-25 00:00:00
abstract：:The phosphoramidate of CCdAp and puromycin (CCdApPuro) is a potent inhibitor of ribosomal peptidyl transferase, as assayed by the fragment reaction. Inhibition is competitive at the ribosomal A-site. CCdApPuro protects P-site-associated bases in the peptidyl transferase loop region of 23S rRNA from carbodiimide modifi...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00002a001
更新日期：1995-01-17 00:00:00
abstract：:The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00292a003
更新日期：1983-11-08 00:00:00
abstract：:Transfection of cos-1 cells with either prostaglandin endoperoxide H synthase-1 (PGHS-1) or -2 (PGHS-2) results in a mixed population of cells containing a diverse range of expressed enzyme. The use of fluorescent substrates and antibodies, in conjunction with fluorescence microscopy, provides the means to quantitate ...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00021a034
更新日期：1995-05-30 00:00:00
abstract：:The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relat...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00478a003
更新日期：1990-07-03 00:00:00
abstract：:Efforts toward the development of RNA-based drug leads have been challenging because of the complexity and dynamic nature of RNA structures as therapeutic targets. The transactivation response (TAR) RNA and cognate Tat protein of HIV have long been recognized as promising antiviral targets, and recent works have ident...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/acs.biochem.5b00325
更新日期：2015-06-16 00:00:00
abstract：:We have covalently attached an electron paramagnetic resonance (EPR) spin probe to Cys-670 of the motor domain of ncd (nonclaret disjunctional protein) in order to investigate conformational changes associated with the chemomechanical cycle. Spin-labeling is highly specific and does not affect ncd function as monitore...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi9706881
更新日期：1997-08-12 00:00:00
abstract：:The mechanism of binding of reduced 1-(2-carboxyphenylamino)-1-deoxyribulose 5-phosphate (rCdRP) to two different binding sites on the bifunctional enzyme is determined by kinetic studies, using temperature-jump and stopped-flow equipment with fluorescence detection. Two rapid binding processes and a comparatively slo...
journal_title：Biochemistry
pub_type： 杂志文章
doi：10.1021/bi00593a030
更新日期：1979-12-25 00:00:00
