Floyd Bryant

• Johns Hopkins Bloomberg School of Public Health

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange react...

Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that...

Streptococcus pneumoniae has two paralogous, homotetrameric, single-stranded DNA binding (SSB) proteins, designated SsbA and SsbB. Previous studies demonstrated that SsbA and SsbB have different solution-dependent binding mode preferences with variable DNA binding capacities. The impact of these different binding properties on the assembly of multi...

The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dTn oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of...

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB prote...

The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB. The SsbA protein is similar in size to the well characterized SSB protein from Escherichia coli (SsbEc). The SsbB protein, in contrast, is a smaller protein that is specifically induced during na...

The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nu...

Streptococcus pneumoniae is a naturally transformable bacterium that is able to incorporate DNA from its environment into its own chromosome. This process, known as transformational recombination, is dependent in part on themmsA gene, which encodes a protein having a sequence that is 40% identical to that of the Escherichia coli RecG protein, a jun...

The ATP-dependent three-strand exchange activity of the Streptococcus pneumoniae RecA protein (RecA(Sp)), like that of the Escherichia coliRecA protein (RecA(Ec)), is strongly stimulated by the single-stranded DNA-binding protein (SSB) from either E. coli (SSB(Ec)) or S. pneumoniae (SSB(Sp)). The RecA(Sp) protein differs from the RecA(Ec) protein,...

The ssDNA-dependent NTP hydrolysis activity of the RecA protein was examined using a series of dTn oligomers ranging in size from dT10 to dT2000 as the ssDNA effector. There were three distinct manifestations of the dTn-dependent NTP hydrolysis reaction, depending on the length of the dTn effector that was used. With longer dTn oligomers, NTP hydro...

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a cir...

The Escherichia coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for the RecA protein-promoted three-strand exchange reaction. An open reading frame encoding a protein similar in size and sequence to the E. coli SSB protein has been identified in the Streptococcus pn...

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 6...

The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence ((66)GPESSGKT(73)) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. As part of an investigation of the role of the...

The RecA protein from the Gram-positive bacterium, Bacillus subtilis, has been reported to catalyze dATP hydrolysis and to promote strand exchange in the presence of dATP but to have no ATP hydrolysis or ATP-dependent strand exchange activity (Lovett, C. M., Jr., and Roberts, J. W. (1985) J. Biol. Chem. 260, 3305-3313). The well characterized RecA...

The ATP analog ATPgammaS is a competitive inhibitor of the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction. The degree of inhibition by ATPgammaS, however, changes in a time-dependent manner and is consistent with a two step binding mechanism. In the first step, ATPgammaS binds to the recA-ssDNA complex in a rapid equilibrium step (K...

We recently constructed a mutant recA protein in which His 163 was replaced by a tryptophan residue. The [H163W]recA protein is functionally identical to the wild-type protein, and the Trp163 side chain serves as a fluorescence reporter group for the ATP and ATPgammaS-mediated conformational transitions of the [H163W]recA-ssDNA complex. In this rep...

We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 μM or less required for stabilization of the strand exchange-active conformation. For e...

We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the flu...

Site-directed mutagenesis was used to replace His-163 in the Loop 1 region of the recA protein with a tryptophan residue. The [H163W]recA protein binds single-stranded DNA (ssDNA), catalyzes ssDNA-dependent ATP hydrolysis, and is fully active in the three-strand exchange reaction. In addition, the fluorescence properties of the Trp-163 reporter gro...

We recently described two mutant recA proteins, (G160N)recA and (H163A)recA, which have full single-stranded DNA-dependent ATP hydrolysis activity but which are unable to promote the ATP-dependent strand exchange reaction under standard reaction conditions (pH 7.5). These mutant proteins, however, are able to promote strand exchange at pH 6.0 to 6....

In previous studies it was shown that the mutant RecA proteins, [G160N]RecA and [H163A]RecA, are unable to catalyze ATP-dependent DNA strand exchanges at pH 7.5, but are active at pH 6.0 to 6.8. Here, we have used electron microscopy to follow the assembly of these mutant proteins onto single-stranded DNA at pH 7.5 and pH 6.2. In the absence of ATP...

The ATP hydrolysis activity of DNA helicase II from Escherichia coli was examined in the presence of linear single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). In the presence of ssDNA, the ATP hydrolysis reaction followed a linear time course until the ATP was depleted. In the presence of dsDNA, in contrast, there was a kinetic lag...

We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine. Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties. Both the [...

The E. coli rec A protein displays a DNA-dependent ATPase activity and is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. Previous work in this laboratory (K. L. Menge and F. R. Bryant, Biochemistry 31, 5151–5157) demonstrated that the structurally related nucleotide triphosphates (NTP), ATP, purine...

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM...

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts...

We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue. The new [Ala-163]recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein. Despite being proficient in ssDNA-dependent ATP hydrolysis, the [Ala-...

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 t...

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 mu...

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. T...

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 micro...

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 approximatel...

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-...

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 th...

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 i...

Excerpt The RecA protein is indispensable for homologous recombination in Escherichia coli (Dressler and Potter 1982). Our aim is to discover its mechanism. A model reaction that has been particularly useful in our analysis has been the exchange of strands between a single-stranded (+) DNA (ssDNA) circle and its homologous duplex (Cox and Lehman 19...

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 addi...

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 su...

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 gr...

The SP diastereomer of adenosine 5'-O-(1-thiodiphosphate) (ADPαS) 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 ADPaS(5P) is present but is greatly act...

A methodology is outlined for examining the stereochemical course at phosphorus of enzyme catalyzed reactions at the phosphodiester level utilizing phosphorothioates. Experiments with intestinal and venom phosphodiesterases that catalyze the formation of 5′-phosphonucleotides and poly-nucleotide phosphorylase that catalyzes the de novo polymerizati...

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 a...