Search

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 +/- 20 bp min-1 at 37 degrees C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 degrees C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 +/- 1.1 kcal mole-1 for DNA strand exchange, and 14.4 +/- 1.4 kcal mole-1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min-1) is related to the kcat for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

Protein n, essential in forming the primosome for the in vitro conversion of phi X174 single-stranded (SS) DNA to the duplex replicative form (RF), has been purified about 5000-fold to near homogeneity from Escherichia coli. Protein n is heat- and acid-resistant and N-ethyl-maleimide-sensitive. It appears to be a dimer of 12,000 (+/- 2000)-dalton polypeptides. About 80 molecules of protein n are present/cell. Protein n binding to phi X SS DNA depends on the presence of single-strand binding protein (SSB). This requirement for SSB reflects a direct interaction of protein n and SSB. About 30 protein n monomers can be bound to an SSB-coated circle. However, in forming the primosome on an SSB-coated phi X circle, an input of only 2-3 protein n monomers is required and 1 monomer bound/circle. Retention of this low level of protein n on SSB-coated phi X SS DNA is dependent upon protein n', a DNA-dependent ATPase (dATPase) that guides primosome assembly. This single protein n monomer is retained in the assembled primosome, which is conserved on the completed parental RF and participates in the next stage of the replicative cycle, production of progeny RF.

The rate of renaturation of denatured, covalently closed, circular DNA (form Id DNA) of the phi X174 replicative form has been investigated as a function of pH, temperature, and ionic strength. The rate at a constant temperature is a sharply peaked function of pH in the range of pH 9 to 12. The position on the pH scale of the maximum rate decreases as the temperature is increased and as the ionic strength is increased. The kinetic course of renaturation is pseudo-first order: it is independent of DNA concentration, but falls off in rate from a first order relationship as the reaction proceeds. The rate of renaturation depends critically on the temperature at which the denaturation is carried out. Form Id, prepared at an alkaline pH at 0 degrees C, renatures from 5 to more than 100 times more rapidly than that similarly prepared at 50 degrees C. Both the heterogeneity in rate and the effect of the temperature of denaturation depend, in part, on the degree of supercoiling of the form I DNA from which the form Id is prepared. However, it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction. The renaturation rate was determined by neutralization of the alkaline reaction and analytical ultracentrifugal analysis of the amounts of forms I and Id. The nature of the proximate renatured species at the temperature and alkaline pH of renaturation was investigated by spectrophotometric titration and analytical ultracentrifugation. It is concluded that the proximate species are the same as the intermediate species defined by an alkaline sedimentation titration of the kind first done by Vinograd et al. ((1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1104-1111). Observations are included on the buoyant density of form Id and on depurination of DNA at alkaline pH values and high temperatures.

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.

The effect of the DNA sequence of a template on the progress of DNA polymerase alpha was determined at the resolution of single nucleotides by using single-stranded, circular phi X174 DNA as a template and unique phi X 174 ONA fragments terminally labeled at their 3'-ends as primers. Therefore, the amount of radioactivity observed was always proportional to the number and not the length of nascent DNA chains. Extension of a primer by alpha-polymerase revealed that 3'-ends of nascent DNA chains accumulated at specific arrest sites consisting of GC-rich sequences of 1-8 bases distributed nonuniformly along the template with intervening sequences of 0-140 bases. The precise location and composition of these sites were determined by DNA sequencing methods, but a consensus sequence was not detected. However, the same pattern of arrest sites recognized by DNA polymerase alpha from CV-1 cells also arrested alpha-polymerase from HeLa cells, calf thymus tissue, and phage T4 DNA polymerase. An exception was DNA polymerase I from Escherichia coli which was insensitive to arrest signals. Analysis of recombinant DNA templates containing phi X174 DNA inserted into M13 DNA revealed that arrest sites were defined exclusively by those sequences within 24 bases upstream and 140 bases downstream of the arrest point; long range interactions between template sequences were not involved. One of the strongest arrest sites was located in the first two nucleotides at the base of the stem on the primer-proximal side of a stable hairpin structure, suggesting that such structures arrest the polymerase. However, only 28% of all arrest site nucleotides were found in this position; the rest were up to 25 bases upstream from any computer-predicted hairpin. Therefore, all arrest sites cannot be defined by secondary structure alone, although proximity to secondary structures may amplify normal variations in the rate of DNA elongation caused by primary sequences.

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition. The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.

Protein n', an enzyme essential for in vitro conversion of single-stranded phiX174 DNA to the duplex replicative form, has been purified about 16,000-fold from Escherichia coli. The enzyme is a single polypeptide chain with a native molecular weight of 76,000; about 70 enzyme molecules are present in an E. coli cell. Nearly homogeneous preparations display an ATPase (dATPase) activity which depends on a unique sequence in the phiX174 DNA. Replicative activity of n' protein and its phiX174 DNA-dependent ATPase activity were present in a constant ratio during the latter stages of purification, upon sedimentation in a glycerol gradient, and during heat inactivation. Further studies of the properties of protein n' are presented in a succeeding paper.

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.

The contribution of proofreading to the fidelity by which Escherichia coli DNA polymerase I copies natural DNA has been analyzed by two independent criteria. With phi X174 am 3 DNA as a template, there is approximately a 25-fold increase in noncomplementary base substitutions at position 587 when the concentration of the next correct nucleotide, dATP, is increased. Sequence analysis indicates that the mistakes represent misincorporation of C in place of T at position 587. This mutagenic response is presumed to result from a decrease in the probability of excision by the 3' leads to 5' exonuclease of Pol I and is considered within the context of current theories on proofreading. No enhanced mutagenicity is observed with avian myeloblastosis virus DNA polymerase, which lacks a 3' leads to 5' exonuclease. Using a second approach, an enhancement in mutagenesis as large as 30-fold is observed to result from the addition of deoxynucleoside monophosphates to the Pol I reaction. This mutagenicity occurs with any of the four deoxynucleoside monophosphates and is independent of a significant inhibition of DNA synthesis, thus supporting proofreading models in which sites of excision and incorporation are independent. The results of both approaches suggest that the exonucleolytic activity of Pol I can increase fidelity by approximately 30-fold on natural DNA, a value much higher than previous estimates with polynucleotide templates. The effect of the next correct nucleotide in decreasing accuracy provides an in vitro probe for screening eukaryotic cells for putative proofreading functions.

A defined region of the viral (+) strand of phi X174 and of each strand of pBR322 DNA serves as an effector for the ATPase activity of replication factor Y from Escherichia coli. These loci can also function as complementary strand origins of DNA replication in a single-stranded circular leads to replicative form pathway whose protein requirements are characteristic of phi X174 DNA. Despite this functional similarity, these three sites possess no extensive sequence homology. To uncover a possible common structural determinant, factor Y recognition sequences were treated with pancreatic DNase or dimethyl sulfate in the presence and absence of this replication protein. When factor Y was present, the action of the nuclease was altered in a similar manner on each of the three templates, indicating that factor Y was bound to the entire length of its effector site. Factor Y-mediated modification of the dimethyl sulfate methylation patterns gave evidence of specific, tight protein-DNA contacts. Protection maps, devised by plotting the results of the methylation and footprinting experiments on duplex structures, suggest that tertiary interactions are either involved in the formation of a factor Y effector site or are induced by the binding of the protein.

The specificity of cleavage of Bam HI is altered in the presence of hydrophobic reagents, such as glycerol and M2SO. The enzyme with altered specificity, designated Bam HI.1, generated digestion patterns of various DNAs, which were distinct from those generated by Bam HI. Cleavage sites recognized in phiX174 RF DNA in the presence of these hydrophobic reagents are not related to the Bam HI palindrome. Bam HI.1 appears to be an endogenous form of Bam HI that can be expressed by altering the hydrophobicity of the reaction.

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.

Conditions which result in DNA strand breakage by the rat liver DNA nicking-closing enzyme lead to the covalent attachment of the 3'-end of the broken strand to the enzyme. Treatment of this complex with pancreatic DNase leaves a residue of 17 +/- 8 nucleotide phosphates still attached to the enzyme. Subsequent nuclease P1 treatment removes all but 2 +/- 1 phosphate residues. Using nuclease P1-treated complexes which had been labeled in the DNA with 32P, the stability of the protein-DNA linkage was studied. The linkage is stable to acid, base, neutral and acidic hydroxylamine, and neutral I2. This pattern of stability rules out essentially all of the possible DNA-protein linkages except for a linkage involving a phosphodiester bond to the amino acid tyrosine. After acid hydrolysis of the 32P-labeled complexes, label was found to be associated with O4-phosphotyrosine, providing a direct demonstration that tyrosine is the amino acid to which the end of the DNA chain is attached.

Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated. The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA. The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used. Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized. Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold. Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from the DNA, which could then reinitiate replication at other available primer templates. Based on these observations a model for SOS-induced UV mutagenesis is proposed.

It has been shown previously (Polisky, B., Green, P., Garfin, D. E., McCarthy, B. J., Goodman, H. M., and Boyer, H. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3310-3314; Hsu, M., and Berg, P. (1978) Biochemistry 17, 131-138) that the cleavage sequence specificity of Eco RI endonuclease can be "relaxed" by various means. In this paper this phenomenon is explored in detail, in order to obtain further insight into the nature and selectivity of sequence recognition patterns between proteins and double-stranded nucleic acids. Using conditions of low ionic strength and alkaline pH, we have mapped the positions of potentially cleavable sites in the (completely sequenced) replicative form of the bacteriophage phi X174 genome, and have deduced their sequence. The time course of digestion of phi X174 DNA suggests that double-stranded sequences reading GGATTT, AAATTT, GAATTT, and GAATTA (only "top" strands, written 5' leads to 3', are shown) are cleaved readily under these conditions, while sequences reading CAATTN (N = A, T, G) resist attack. Cleavages at (at least) the more labile sites result in cohesive ends that are religatable. End group analysis of cleaved phi X174 DNA fragments indicates the presence of a 5'-terminal adenine residue on most of the fragments; some fragments may carry a 5'-terminal guanine residue, consistent with the cleavage site sequences suggested above. Addition of Mn2+ to cleavage reactions carried out at moderate salt concentrations and near-neutral pH induces the same pattern of cleavage seen at low ionic strength and alkaline pH. These results are combined with those from other studies, and are interpreted in terms of a model for the site-specific interaction of the Eco RI endonuclease with its substrate, considering both the effects of changes in DNA sequence and of environmental alterations. The resulting model is compared with data developed on similar grounds for Eco RI methylase (see Woodbury, C. P., Downey, R. L., and von Hippel, P. H. (1980) J. Biol. Chem. 255, 11526-11533), and attempts are made to define both common and differing molecular facets of the DNA recognition specificity of these companion (but genetically distinct) enzymes.

We propose a model to describe the frequencies of site-specific base substitution errors by DNA polymerase. In the model, nucleotide misinsertion frequencies are determined by 5'-nearest-neighbor base stacking and 3'-exonucleolytic proofreading efficiencies are governed by the relative proportions of G . C base pairs in the region surrounding the misinserted nucleotide. The model is used to analyze the frequency of replacing dAMP by 2-aminopurine deoxyribonucleotide with purified bacteriophage T4 L141 antimutator DNA polymerase at 57 sites on phi X174 DNA (Pless, R. C., and Bessman, M.J. (1983) Biochemistry 22, 4905-4915). A linear least-squares fit yields a correlation coefficient of 0.83 and a standard deviation of 2.8% between predicted and observed results. Four to five base pairs on each side of the 2-aminopurine incorporation site, approximately one double-helical turn, are found to exert a maximal influence on proofreading. Thermal melting data on native and synthetic DNA are used to deduce base-stacking energies for nearest-neighbor doublets including those involving 2-aminopurine. The inclusion of base-stacking energies in the model calculations leads to predictions similar to those based on the use of empirical parameters for individual base pairs.