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C C Richardson

Publications and source records attributed to C C Richardson.

At least 109 records · Page 6Linked to original sources
PubMed1983

DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7.

The gene 4 protein of bacteriophage T7 is both a primase and a helicase. In this paper, we present a detailed description of a third activity, single-stranded DNA-dependent nucleoside 5'-triphosphate hydrolysis, and show that this activity is coupled to the unidirectional translocation of the gene 4 protein on single-stranded DNA (Tabor, S., and Richardson, C.C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 205-209). The competitive inhibitor of NTP hydrolysis, beta, gamma-methylene dTTP, is also a potent inhibitor of gene 4 protein-dependent, RNA-primed DNA synthesis; inhibition is not due to a direct inhibition of T7 DNA polymerase or RNA primer synthesis. We conclude that the energy derived from the hydrolysis of NTPs by the gene 4 protein is required for translocation of the protein to primase recognition sites. Measurement of the rates of hydrolysis of NTPs using a variety of DNAs of known structure and length support the unidirectional translocation of the gene 4 protein on single-stranded DNA. Duplex DNA, RNA, and single-stranded DNA coated with single-stranded DNA-binding protein do not serve as effectors for the nucleoside triphosphatase of the gene 4 protein. Kinetic data suggest that the gene 4 protein does not remain bound to newly synthesized oligoribonucleotide primers but continues to search for other primase recognition sites. Although all the predominant naturally occurring NTPs except rCTP are hydrolyzed by the gene 4 protein, the enzyme shows specificity for dTTP with a Km of 0.4 mM. In the accompanying paper (Matson, S.W., Tabor, S., and Richardson, C.C. (1983) J. Biol. Chem. 258, 14017-14024), we show that the hydrolysis of NTPs is also required for the protein to function as a helicase in duplex regions of DNA.

Adenosine Triphosphatases↗
PubMed1983

The gene 4 protein of bacteriophage T7. Characterization of helicase activity.

Gene 4 protein of bacteriophage T7 is a multifunctional enzyme that both stimulates T7 DNA polymerase during leading strand synthesis, and synthesizes RNA primers that initiate lagging strand synthesis. Both activities are dependent on the ability of the gene 4 protein to translocate unidirectionally (5' to 3') along single-stranded DNA (Tabor, S., and Richardson, C.C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 205-209), a reaction that is coupled to the hydrolysis of nucleoside 5'-triphosphates. In this paper, we show that gene 4 protein, in the absence of other proteins, is a helicase, an activity previously inferred from its ability to stimulate T7 DNA polymerase on duplex DNA. We have designed a DNA substrate for use in characterizing the helicase activity which consists of a short DNA fragment bearing a single-stranded 3'-tail when annealed to circular, single-stranded M13 DNA. With such a DNA substrate, the gene 4 protein can disrupt the helical structure of a 96-nucleotide-long fragment, resulting in its displacement from the circle. Helicase activity requires a long stretch of at least 17 nucleotides of single-stranded DNA on the 5'-side of the duplex to be unwound. In addition, helicase activity is not observed unless a short (greater than 6 nucleotides) single-stranded 3'-tail is present. The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate. The inhibitor of nucleoside triphosphate hydrolysis, beta, gamma-methylene dTTP, is an effective inhibitor of helicase activity. Based on these results, we propose a model for the action of the gene 4 protein at a replication fork.

DNA Helicases↗
PubMed1983

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase.

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.

Base Sequence↗
PubMed1983

Two forms of the DNA polymerase of bacteriophage T7.

The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.

DNA Replication↗
PubMed1983

A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein.

This paper describes the construction of a DNA molecule containing a topologically stable structure that simulates a replication fork. This preformed DNA molecule is a circular duplex of 7.2 X 10(3) base pairs (M13mp6 DNA) from which arises, at a unique BamHI recognition site, a noncomplementary 5'-phosphoryl-terminated single strand of 237 nucleotides (SV40 DNA). This structure has two experimental attributes. 1) Templates for both leading and lagging strand synthesis exist as stable structures prior to any DNA synthesis. 2) DNA synthesis creates a cleavage site for the restriction endonuclease BamHI. Form I of T7 DNA polymerase, alone, catalyzes limited DNA synthesis at the preformed replication fork whereas Form II, alone, polymerizes less than 5 nucleotides. However, when T7 gene 4 protein is present, Form II of T7 DNA polymerase catalyzes rapid and extensive synthesis via a rolling circle mode. Kinetic analysis of this synthesis reveals that the fork moves at a rate of 300 bases/s at 30 degrees C. We conclude that the T7 gene 4 protein requires a single-stranded DNA binding site from which point it translocates to the replication fork where it functions as a helicase. The phage T4 DNA polymerase catalyzes DNA synthesis at this preformed replication fork in the presence of gene 4 protein, but the amount of DNA synthesized is less that 3% of the amount synthesized by the combination of Form II of T7 DNA polymerase and gene 4 protein. We conclude that T7 DNA polymerase and T7 gene 4 protein interact specifically during DNA synthesis at a replication fork.

DNA Replication↗
PubMed1983

Bacteriophage T7 DNA replication. Synthesis of lagging strands in a reconstituted system using purified proteins.

Replication of the lagging strand of bacteriophage T7 DNA occurs in a discontinuous fashion that requires RNA-primed DNA synthesis, the removal of the RNA primers, the replacement of the ribonucleotides with deoxyribonucleotides, and the covalent joining of adjacent DNA fragments. We have examined each of these steps as well as the whole process through the use of model substrates and partial reactions using purified proteins. Tetraribonucleotides (pppACCC or pppACCA), synthesized by the T7 gene 4 protein on single-stranded DNA, are used as primers by T7 DNA polymerase to yield RNA-terminated DNA fragments. The removal of the RNA primers is catalyzed by the 5' to 3' hydrolytic activities of either Escherichia coli DNA polymerase I or the T7 gene 6 exonuclease. The products of hydrolysis are pppApC, ATP, and nucleoside 5'-monophosphates or ATP and nucleoside 5'-monophosphates, respectively. The requirement for DNA synthesis to fill the gap between adjacent DNA fragments can be fulfilled by Form II of T7 DNA polymerase but not by Form I. DNA synthesis catalyzed by Form II of T7 DNA polymerase eliminates gaps to create a substrate for DNA ligase whereas strand displacement synthesis catalyzed by Form I creates an aberrant structure that cannot be joined. Either the host or phage DNA ligase can effect the final covalent joining. All steps in the replication of a lagging strand have been coupled in a model system that catalyzes the formation of covalently closed, circular, double-stranded DNA molecules using single-stranded viral DNA as template. A combination of four bacteriophage proteins, gene 4 protein, Form II of T7 DNA polymerase, gene 6 exonuclease, and DNA ligase, can accomplish this overall reaction.

DNA Replication↗
PubMed1981

Processing of mRNA by ribonuclease III regulates expression of gene 1.2 of bacteriophage T7.

A bacteriophage T7 mutation, HS9, is phenotypically defective in gene 1.2, although it maps outside the gene. The single nucleotide change responsible for the HS9 mutation lies within the RNAase III recognition site immediately following gene 1.2. This RNAase III recognition site, responsible for the processing of the mRNA encoding genes 1.1 and 1.2, contains two cleavage sites, separated by 29 bases. The HS9 mutation prevents cutting by RNAase III at one site in vitro, yielding a mRNA containing an additional 29 bases at its 3' end. The ten second-site reversion mutations of HS9 are all located in the RNAase III recognition site and either restore or eliminate cutting at both sites. RNAase III mutants of Escherichia coli phenotypically suppress the HS9 mutation. We propose that the extra 29 bases at the 3' end of the mRNA hybridize to the ribosome-binding site of gene 1.1; gene 1.1 immediately precedes gene 1.2 on the same mRNA molecule. Such hybridization prevents the initiation of translation of this mRNA containing gene 1.1. A strong polar effect represses the translation of gene 1.2.

Base Sequence↗
PubMed1981

Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7.

The gene 4 protein of bacteriophage T7 recognizes specific sequences on single-stranded DNA and then catalyzes the synthesis of tetraribonucleotide primers complementary to the template. With phi X174 DNA as a template, the gene 4 protein enables T7 DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) to initiate DNA synthesis at 13 major sites. DNA sequence analysis of the 5' termini of the newly synthesized DNA shows the predominant recognition sequences for the gene 4 protein to be 3'-C-T-G-G-G-5' or 3'-C-T-G-G-T-5'; the products of synthesis at these sites are RNA primers having the sequences pppA-C-C-C or pppA-C-C-A. The gene 4 protein can also synthesize primers at the sequences 3'-C-T-G-G-AC-5' and 3'-C-T-G-T-N-5', although these sites are used less than 10% as frequently as the predominant sites. Comparison of the utilization of primer sites suggests that the gene 4 protein binds randomly to single-stranded DNA and then translocates along the DNA in a unidirectional 5'-to-3' direction with regard to the DNA strand in search of recognition sequences. Models are presented for the role of the gene 4 protein in the initiation of lagging-strand synthesis and in the initiation of DNA replication at the origin.

Bacteriophage phi X 174↗
PubMed1981

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.

The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.

Base Sequence↗
PubMed1981

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.

Chromosome Mapping↗
PubMed1980

Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication.

Deletion mutants of bacteriophage T7 have been used to identify and to map, by electron microscopy, the origins of T7 DNA replication. The primary origin of phage T7 DNA replication lies within a 100-base-pair region located 14.75-15.0% of the distance from the genetic left end of the DNA molecule. T7 phage whose DNA contains a deletion of this region initiate replication at secondary origins, the predominant one of which is located at a distance approximately 4% from the left end of the molecule.

Chromosome Deletion↗
PubMed1980

Nucleotide sequence of the primary origin of bacteriophage T7 DNA replication: relationship to adjacent genes and regulatory elements.

The 682-base-pair nucleotide sequence between positions 14.45 and 16.15 on the bacteriophage T7 DNA molecule has been determined. We can identify not only the sequence of the primary origin of DNA replication but also the termination of gene 1, all of genes 1.1 and 1.2, the start of gene 1.3, and a number of regulatory sequences. The endpoints of four deletion mutations that extend into this region have been determined. These mutations are inferred to have arisen by recombination between short homologous sequences, three of which ar T7 RNA polymerase promoters. The base changes of four point mutations in gene 1.2 have been identified. The sequence essential for initiation at the primary origin is located between the left endpoints of the two deletions D2 and D303. Sequence analysis of these mutants assigns the primary origin to a 129-base-pair segment between positions 14.73 and 15.05. This intergenic segment is A+T-rich (75%) and contains a single T7 gene 4 protein recognition site; it is preceded by two tandem T7 RNA polymerase promoters. A model for initiation of T7 DNA replication is presented.

Base Sequence↗
PubMed1979

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.

DNA Helicases↗
PubMed1979

Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.

DNA-Directed DNA Polymerase↗
PubMed1979

Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7.

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze DNA synthesis on duplex DNA templates. Synthesis is initiated at nicks in the DNA template, and this leading strand synthesis results in displacement of one of the parental strands. In the presence of ribonucleoside 5'-triphosphates the gene 4 protein catalyzes the synthesis of oligoribonucleotide primers on the displaced single strand, and their extension by T7 dna polymerase accounts for lagging strand synthesis. Since all the oligoribonucleotide primers bear adenosine 5'-triphosphate residues at their 5' termini, [gamma 32P]ATP is incorporated specifically into the product molecule, thus providing a rapid and sensitive assay for the synthesis of the RNA primers. Both primer synthesis and DNA synthesis are stimulated 3- to 5-fold by the presence of either Escherichia coli or T7 helix-destabilizing protein (DNA binding protein). ATP and CTP together fully satisfy the requirement for rNTPs and provide maximum synthesis of primers and DNA. Provided that T7 DNA polymerase is present, RNA-primed DNA synthesis occurs on either duplex or single-stranded DNA templates and to equal extents on either strand of T7 DNA. No primer-directed DNA synthesis occurs on poly(dT) or poly(dG) templates, indicating that synthesis of primers is template-directed.

DNA Helicases↗
PubMed1979

Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.

DNA Helicases↗