Biomedical subjects

C C Richardson

Publications and source records attributed to C C Richardson.

At least 37 records · Page 2Linked to original sources

PubMed1998

Enhanced binding of azidothymidine-resistant human immunodeficiency virus 1 reverse transcriptase to the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer.

Human immunodeficiency virus type 1 is resistant to 3'-azido-3'-deoxythymidine (AZT) when four amino acid substitutions (D67N, K70R, T215F, and K219Q) are present simultaneously in its reverse transcriptase. Wild-type and AZT-resistant reverse transcriptases show identical binding to a 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer/RNA template. On DNA templates, the equilibrium dissociation constant (KD) for primer/template and AZT-resistant reverse transcriptase (RT) (KD = 4.1 nM) is similar to that of the wild-type enzyme (KD = 6.2 nM). However, koff is 4-25-fold lower for the AZT-resistant enzyme than for the wild-type enzyme, depending on the nucleotide and the template. The kinetic decay of a wild-type RT/primer/AZTMP-terminated DNA template complex is biphasic. Seventy percent of the initial complex decays with a rate constant greater than 0.05 s-1, and 30% with a rate constant of 0.0017 s-1. Decay of an AZT-resistant RT/AZTMP-terminated primer/DNA template complex is monophasic, with a rate constant of 0.0018 s-1. The last two nucleotides at the 3' end of the AZTMP-terminated DNA primer in complex with AZT-resistant RT, but not wild-type RT, and a DNA template are protected from exonuclease digestion, suggesting that enhanced binding of the 3' end of the AZTMP-terminated DNA primer to reverse transcriptase is involved in the mechanism of AZT resistance by human immunodeficiency virus type 1.

Cross-Linking Reagents↗

PubMed1998

Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions.

The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination. Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase. A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro. The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo. To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E. coli SSB protein or T4 gene 32 protein. Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7. The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities. In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E. coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7. We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.

Amino Acid Sequence↗

PubMed1998

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

Bacteriophage T7↗

PubMed1998

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

Bacteriophage M13↗

PubMed1998

Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution.

DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6). Here we present a 2.2 A crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

Bacteriophage T7↗

PubMed1998

Coordinated leading and lagging strand DNA synthesis on a minicircular template.

The coordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand so that both strands can be synthesized in the same direction. We have constructed a minicircle with a replication fork that permits an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand. The replisome consisting of bacteriophage T7 DNA polymerase, helicase, primase, and single-stranded DNA-binding protein mediates coordinated replication. The criteria for coordination are fulfilled: (1) a replication loop is formed, (2) leading and lagging strand synthesis are coupled, (3) the lagging strand polymerase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated. T7 single-stranded DNA-binding protein is essential for coordination.

Bacteriophage T7↗

PubMed1997

Binding of RNA template to a complex of HIV-1 reverse transcriptase/primer/template.

HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.

Binding Sites↗

PubMed1997

The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase.

The gene 4 proteins of bacteriophage T7 provide both primase and helicase activities at the replication fork. Efficient DNA replication requires that the functions of the gene 4 protein be coordinated with the movement of the T7 DNA polymerase. We show that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis. The carboxyl terminus of the gene 4 protein is highly acidic: of the 17 carboxyl-terminal amino acids 7 are negatively charged. Deletion of the coding region for these 17 residues results in a gene 4 protein that cannot support the growth of T7 phage. The purified mutant gene 4 protein has wild-type levels of both helicase and primase activities; however, DNA synthesis catalyzed by T7 DNA polymerase on a duplex DNA substrate is stimulated by this mutant protein to only about 5% of the level of synthesis obtained with wild-type protein. The mutant gene 4 protein can form hexamers and bind single-stranded DNA, but as determined by native PAGE analysis, the protein cannot form a stable complex with the DNA polymerase. The mutant gene 4 protein can prime DNA synthesis normally, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved. These findings have implications for the mechanisms coupling leading and lagging strand DNA synthesis at the T7 replication fork.

Amino Acid Sequence↗

PubMed1997

Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites.

The 63-kDa gene 4 primase of bacteriophage T7 recognizes a core trinucleotide sequence, 5'-GTC-3', on single-stranded DNA at which it catalyzes the synthesis of the ribodinucleotide pppAC. The dinucleotide is extended to a tetranucleotide primer at the sites 5'-(G/T)GGTC-3' and 5'-GTGTC-3'. In the presence of T7 primase, T7 DNA polymerase extends the synthetic ribotetranucleotide pACCA (1 microM), but not pCACA, on M13 DNA templates. The reaction is specific for T7 DNA polymerase and depends on dTTP and translocation of the gene 4 protein. T7 primase extends the dinucleotide AC and trinucleotide ACC to ACCC in the presence of CTP and an appropriate template, whereas other dinucleotides are extended less efficiently; the deoxyribodinucleotide dAC is not extended. The Cys4 zinc motif of the primase is essential for extension of the dinucleotides. The 5'-cryptic cytidine of the recognition sequence is essential for extension of the dinucleotide AC to tri- and tetranucleotides. At a preformed replication fork, the dinucleotide AC provides for primer synthesis on the lagging strand. The synthesis of all Okazaki fragments is initiated by primers arising from the recognition sequence 5'-GGGTC-3'; none arise at an adjacent 5'-GGGTT-3' sequence. If ADP or AMP replaces ATP in the primase reaction, primers terminating in di- or monophosphate, respectively, are synthesized.

Adenosine Diphosphate↗

PubMed1997

Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions.

In bacteriophage T7 the gene 2.5 single-stranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.

Bacteriophage T7↗

PubMed1997

Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration.

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.

Bacteriophage T4↗

PubMed1997

Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization.

T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA.

Bacteriophage T7↗

PubMed1997

Template recognition and ribonucleotide specificity of the DNA primase of bacteriophage T7.

The 63-kDa gene 4 DNA primase of phage T7 catalyzes the synthesis of oligoribonucleotides on single-stranded DNA templates. At the sequence, 5'-GTC-3', the primase synthesizes the dinucleotide pppAC; the cytidine residue of the recognition sequence is cryptic. Only tetraribonucleotides function as primers, but the specificity for the third and fourth position is not as stringent with a preference of CMP > AMP >> UMP > GMP. The predominant recognition sites on M13 DNA are 5'-(G/T)GGTC-3' and 5'-GTGTC-3'. Synthesis is usually limited to tetranucleotides, but T7 primase can synthesize longer oligoribonucleotides on templates containing long stretches of guanosine residues 5' to the recognition sequence. The specificity beyond the first two positions of the primer increases as the length of the template on the 3'-side of 5'-GTC-3' increases. On an oligonucleotide having 20 3'-flanking cytidine residues GMP is incorporated at the third position; incorporation is reduced 4-fold when the flanking sequence reaches 65 residues, and little is incorporated on M13 templates. The presence of the 56-kDa gene 4 helicase decreases the incorporation of GMP on long templates. We propose that pausing is required for the incorporation of less preferred nucleotides and that pausing is decreased by the ability of the primase to translocate 5' to 3' on templates having long 3'-flanking sequences.

Bacteriophage T7↗

PubMed1997

The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I.

Bacteriophage T7 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I. However, in vivo, E. coli DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome. In accord with these roles, T7 DNA polymerase is highly processive while E. coli DNA polymerase I has low processivity. The high processivity of T7 DNA polymerase is achieved through tight binding to its processivity factor, E. coli thioredoxin. We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction. Insertion of this domain into the homologous site in E. coli DNA polymerase I results in a dramatic increase in the processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.

Bacteriophage T7↗

PubMed1996

Structural and functional organization of the DNA polymerase of bacteriophage T7.

The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.

Amino Acid Sequence↗

PubMed1996

Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.

Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774-9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3' to 5' exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.

Bacterial Proteins↗

PubMed1996

The role of the zinc motif in sequence recognition by DNA primases.

The DNA primase of bacteriophage T7 has a zinc-binding motif that is essential for the recognition of the sequence 3'-CTG-5'. The T7 primase also catalyzes helicase activity, a reaction coupled to nucleotide hydrolysis. We have replaced the zinc motif of the T7 primase with those found in the gene 61 primase of phage T4 and the DnaG primase of Escherichia coli. The T4 and E. coli primases recognize the sequences 3'-T(C/T)G-5' and 3'-GTC-5', respectively. Both chimeric proteins can partially replace T7 primase in vivo. The two chimeric primases catalyze the synthesis of oligoribonucleotides albeit at a reduced rate and DNA dependent dTTPase activity is reduced by 3-10-fold. Both chimeric proteins recognize 3'-(A/G)CG-5' sites on single-stranded DNA, sites that differ from those recognized by the T7, T4, or E. coli primases, indicating that the zinc motif is only one determinant in site-specific recognition.

Amino Acid Sequence↗

PubMed1996

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.

Bacteriophage T7↗