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F R Bryant

Publications and source records attributed to F R Bryant.

32 records · Page 2Linked to original sources
PubMed1990

An obligatory pH-mediated isomerization on the [Asn-160]recA protein-promoted DNA strand exchange reaction pathway.

We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F. R. (1988) J. Biol. Chem. 263, 8716-8723). Although the [Asn-160]recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP). We have found that the [Asn-160]recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH. The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the [Asn-160]recA protein. This ATP-dependent isomerization is characterized by the conversion of the [Asn-160]recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein. In contrast to the pronounced pH sensitivity of the [Asn-160]recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4. These results show that the [Asn-160] mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the [Asn-160]recA protein (or the [Asn-160]recA-ssDNA complex) relieves this mechanistic defect. Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the [Asn-160]recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism.

Adenosine Triphosphatases↗
PubMed1989

Kinetic modeling of the recA protein promoted renaturation of complementary DNA strands.

Quantitative agarose gel assays reveal that the recA protein promoted renaturation of complementary DNA strands (phi X DNA) proceeds in two stages. The first stage results in the formation of unit-length duplex DNA as well as a distribution of other products ("initial products"). In the second stage, the initial products are converted to complex multipaired DNA structures ("network DNA"). In the presence of ATP, the initial products are formed within 2 min and are then rapidly converted to network DNA. In the absence of ATP, the initial products are formed nearly as fast as with ATP present, but they are converted to network DNA at a much lower rate. The time-dependent formation of initial products and network DNA from complementary single strands for both the ATP-stimulated and ATP-independent reactions can be modeled by using a simple two-step sequential kinetic scheme. This model indicates that the primary effect of ATP in the recA protein promoted renaturation reaction is not on the initial pairing step (which leads to the formation of initial products) but rather is to increase the rate at which subsequent pairing events can occur.

Adenosine Triphosphate↗
PubMed1988

Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity.

The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue. The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein. Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2. To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue. The new [Asn160]recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein. This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain. Although it is an active single-stranded DNA-dependent ATPase, the [Asn160]recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro. Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the [Asn160]recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E. coli protein. To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the [Asn160]recA protein.

Adenosine Triphosphatases↗
PubMed1988

ATP-stimulated hydrolysis of GTP by RecA protein: kinetic consequences of cooperative RecA protein-ATP interactions.

The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP). At pH 7.5 and 37 degrees C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1. The S0.5 for GTP (750 microM), however, is nearly 20-fold higher than the S0.5 for ATP (45 microM). Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5 for GTP; in the presence of 50 microM ATP, the S0.5 for GTP is reduced from 750 microM to 200 microM. Concentrations of ATP greater than 50 microM result in competitive inhibition of the ATP-activated GTPase activity. Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction. To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.

Adenosine Triphosphate↗
PubMed1986

ATP-independent renaturation of complementary DNA strands by the mutant recA1 protein from Escherichia coli.

In an effort to clarify the requirement for ATP in the recA protein-promoted renaturation of complementary DNA strands, we have analyzed the mutant recA1 protein which lacks single-stranded DNA-dependent ATPase activity at pH 7.5. Like the wild type, the recA1 protein binds to single-stranded DNA with a stoichiometry of one monomer per approximately four nucleotides. However, unlike the wild type, the mutant protein is dissociated from single-stranded DNA in the presence of ATP or ADP. The ATP analogue adenosine 5'-O-3' (thiotriphosphate) appears to stabilize the binding of recA1 protein to single-stranded DNA but does not elicit the stoichiometry of 1 monomer/8 nucleotides or the formation of highly condensed protein-DNA networks that are characteristic of the wild type recA protein in the presence of this analogue. The recA1 protein does not catalyze DNA renaturation in the presence of ATP, consistent with the dissociation of recA1 protein from single-stranded DNA under these conditions. However, it does promote a pattern of Mg2+-dependent renaturation identical to that found for wild type recA protein.

Adenosine Triphosphate↗
PubMed1985

Interaction of the recA protein of Escherichia coli with single-stranded DNA.

The interaction of recA protein with single-stranded (ss) phi X174 DNA has been examined by means of a nuclease protection assay. The stoichiometry of protection was found to be 1 recA monomer/approximately 4 nucleotides of ssDNA both in the absence of a nucleotide cofactor and in the presence of ATP. In contrast, in the presence of adenosine 5'-O-(thiotriphosphate) (ATP gamma S) the stoichiometry was 1 recA monomer/approximately 8 nucleotides. No protection was seen with ADP. In the absence of a nucleotide cofactor, the binding of recA protein to ssDNA was quite stable as judged by equilibration with a challenge DNA (t1/2 approximately 30 min). Addition of ATP stimulated this transfer (t1/2 approximately 3 min) as did ADP (t1/2 approximately 0.2 min). ATP gamma S greatly reduced the rate of equilibration (t1/2 greater than 12 h). Direct visualization of recA X ssDNA complexes at subsaturating recA protein concentrations using electron microscopy revealed individual ssDNA molecules partially covered with recA protein which were converted to highly condensed networks upon addition of ATP gamma S. These results have led to a general model for the interaction of recA protein with ssDNA.

Adenosine Diphosphate↗
PubMed1985

On the mechanism of renaturation of complementary DNA strands by the recA protein of Escherichia coli.

The renaturation of complementary DNA strands by the recA protein of Escherichia coli has been found to exhibit the following features. (i) Optimal renaturation occurs at recA protein levels below that required to saturate the DNA strands; saturating amounts of recA protein significantly reduce the rate of reaction. (ii) The reaction proceeds in the absence of a nucleotide cofactor but is markedly stimulated by ATP in the presence of 10 mM Mg2+. A similar stimulation occurs in the absence of ATP when the Mg2+ concentration is increased from 10 mM to 30-40 mM. (iii) Both the ATP-stimulated and the Mg2+-stimulated reactions follow apparent first-order kinetics. These results, taken together with the known effects of ATP and Mg2+ on the state of aggregation of recA protein, suggest that the association of recA monomers may play an important role in recA protein-promoted DNA renaturation.

Adenosine Triphosphate↗
PubMed1984

Continuous assay for DNA polymerization by light scattering.

The time course of DNA polymerization, catalyzed by DNA polymerase I, can be monitored by following changes in light scattering that result from increases in molecular weight of the polymer. Increases in mass of 10% result in an easily measurable change in light scattering. It has been shown that the light scattering change is correlated with the incorporation of radioactive nucleotide, as measured by the filter binding method. Continuous monitoring of the time dependence of polymerization should prove useful in kinetic analysis of the polymerase reaction and related enzyme-DNA interactions.

DNA↗
PubMed1983

Elementary steps in the DNA polymerase I reaction pathway.

The polymerization reaction catalyzed by Escherichia coli DNA polymerase I (Pol I) has been studied by using the homopolymer template-primer system poly(dA).oligo(dT). Isotope-partitioning experiments indicate that the reaction follows an ordered mechanism in which Pol I first combines with template-primer to form an E.poly complex followed by addition of MgTTP and catalysis. The parameters governing the binding of Pol I to the template-primer are kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, and KD = 2 X 10(-7) M. Efforts to demonstrate the catalytic competence of the binary E.MgTTP complex were unsuccessful. Following initiation of the catalytic cycle, Pol I catalyzes the incorporation of an average of 40-50 TTP molecules into polymer before dissociating from the template-primer. The processive nature of the polymerization reaction as reflected by the isotope-trapping time dependence can be accounted for by a model in which processive synthesis is treated as a simple partitioning between continued polymerization (kcat = 3.8 s-1, 22 degrees C) and dissociation of the enzyme from the template-primer under steady-state conditions (koffss = 0.1 s-1). The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 degrees C) exhibited a pre-steady-state burst consistent with two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k = 4.6 s-1) and the other which follows this step (k = 4.0 s-1). Binding of MgTTP to the E.poly complex was shown to be a rapid equilibrium step by steady-state isotope-partitioning experiments. This suggested that the first rate-determining step may be a first-order isomerization which follows the binding of substrates and precedes bond formation.

DNA Polymerase I↗
PubMed1982

Phosphorothioate substrates for T4 RNA ligase.

Stereochemical details of the T4 RNA ligase reaction mechanism have been delineated by examining the reactivity of phosphorothioate analogues in the adenosine 5'-triphosphate (ATP) dependent and ATP-independent RNA ligase reactions. Only the SP isomer of the diastereomeric dinucleoside thiopyrophosphate, App(s)I, was active as an activated donor substrate in the ATP-independent RNA ligase reaction. The thiophosphodiester linkage in the ligation product, ApApAp(s)I, that is formed by the reaction of App(s)I (SP) with the oligonucleotide acceptor, ApApA, was shown to have the RP configuration. This indicates that phosphodiester bond formation occurs by a direct, nucleophilic displacement of AMP from App(s)I by the 3'-hydroxyl group of ApApA with inversion of configuration at phosphorus. The adenylylated intermediate, App(s)Ap, that is formed from the phosphorothioate donor, p(s)Ap, in the ATP-dependent RNA ligase reaction was shown to have the same stereochemical configuration as is required for the ATP-independent RNA ligase reaction. These results indicate that RNA ligase maintains a preferred chirality at phosphorus through the adenylylation and ligation steps of the reaction mechanism. An unusual result is the accumulation of adenosine cyclic 2',3'-phosphate 5'-phosphorothioate in the ATP-dependent RNA ligase reaction employing the donor p(s)Ap when the acceptor ApApA is present. This observation suggests that there are two distinct but reactive modes for donor molecules.

Adenine Nucleotides↗
PubMed1981

The stereochemical course of thiophosphoryl group transfer catalyzed by T4 polynucleotide kinase.

The stereochemical course of the phosphoryl transfer reaction catalyzed by T4 polynucleotide kinase has been determined using the chiral ATP analog, (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate). T4 polynucleotide kinase catalyzes the transfer of the gamma-thiophosphoryl group of (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate) to the 5'-hydroxyl group of ApA to give the thiophosphorylated dinucleotide adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine. A sample of adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine was subjected to venom phosphodiesterase digestion. The resulting adenosine-5'-[18O]phosphorothioate was shown to have the Rp configuration, thus indicating that the thiophosphoryl transfer reaction occurs with overall inversion of configuration of phosphorus.

Adenine Nucleotides↗
PubMed1981

Stereochemical and kinetic investigation of 32P-labeled inorganic phosphate exchange reaction catalyzed by primer-independent and primer-dependent polynucleotide phosphorylase from Micrococcus luteus.

The SP diastereomer of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) is a substrate for the 32P-labeled inorganic phosphate exchange reaction catalyzed by the T and I forms of polynucleotide phosphorylase. The exchange reaction occurs with retention of configuration. This exchange reaction is very slow when only ADP alpha S(SP) is presented but is greatly activated by dinucleotide primers and ADP alpha S(RP), although the latter is not a substrate for the exchange reaction. Ap(S)A(RP) is an approximately 50% better activator of the exchange than the SP diastereomer. Furthermore, high levels of the ADP alpha S(SP) eliminate the activation by primers and by ADP alpha S(RP). A phosphatase activity is present with the I form of the enzyme which converts ADP alpha S(RP) to AMPS. This activity may be responsible for the formation of the 5'-phosphate end group for de novo polymerization or for the processivity of this reaction.

Adenosine Diphosphate↗
PubMed1979

Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.

Crotalid Venoms↗