Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins

The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves. Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein. However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein. As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E. coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B. M. (1983) Cell 34, 115-123).     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.     

The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase.

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.     

Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme.

The gene encoding the delta' subunit of DNA polymerase III holoenzyme, designated holB, was cloned by a strategy in which peptide sequence was used to derive a DNA hybridization probe. The gene maps to 24.95 centisomes of the chromosome. Sequencing of holB revealed a 1,002-bp open reading frame predicted to produce a 36,936-Da protein. The gene has a ribosome-binding site and promoter that are highly similar to the consensus sequences and is flanked by two potential open reading frames. Protein sequence analysis of delta' revealed a high degree of similarity to the dnaX gene products of Escherichia coli and Bacillus subtilis, including one stretch of 10 identical amino acid residues. A lesser degree of similarity to the gene 44 protein of bacteriophage T4 and the 40-kDa protein of the A1 complex (replication factor C) of HeLa cells was seen. The gene, when placed into a tac promoter-based expression plasmid, directed expression of two proteins of similar size. By immunodetection with anti-holoenzyme immunoglobulin G, both proteins are judged to be products of holB.     

Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates

Sequence-specific pausing occurs during DNA synthesis catalyzed by the bacteriophage T4 DNA polymerase holoenzyme in the presence of the T4 helix destabilizing protein (gene 32 protein). Two of the six strongest pause sites on a double-stranded bacteriophage fd DNA template are in regions where hairpin helices are predicted to form when the DNA is single stranded. However, the other pause sites are in regions that are not obviously involved in secondary structure. The positions of the DNA chain ends produced at one pause site of each type were determined to within +/- 2 nucleotides. At this resolution, a clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions. The addition of the T4 gene 41 protein (a DNA helicase that forms part of the T4 primosome) to the above replication system greatly increases the rate of fork movement and eliminates detectable pausing. In contrast, the addition of the T4 dda protein (a second DNA helicase that increases the rate of fork movement to a similar extent) has no affect on replication fork pausing. This difference could either be due to specific protein-protein interactions formed between the polymerase holoenzyme and the 41 protein or to the highly processive movement of the 41 protein along the displaced DNA strand.     

Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.     

Purification and properties of the Escherichia coli dnaK replication protein

The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).     

Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.

DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.     

The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro

An in vitro system containing the T4 gene 43, 45, 44/62, 32, dda, and uvsX proteins catalyzes DNA synthesis that is dependent on the synapsis step of homologous genetic recombination. The rate of DNA synthesis in this system is highly dependent on the concentration of the uvsX recombinase (a recA-like protein). Here we report the effect of the T4 uvsY protein, a recombination accessory protein, on this reaction. Low concentrations of uvsY protein greatly stimulate DNA synthesis at low concentrations of uvsX protein, but these same concentrations inhibit DNA synthesis at high concentrations of uvsX protein. As a result, the addition of small amounts of uvsY protein lowers the minimum concentration of uvsX protein needed for the reaction 8-fold, and it lowers the uvsX protein concentration for maximum activity 4-fold. The uvsY protein can affect either the initiation or elongation phase of DNA synthesis, depending on the concentration of uvsX protein present. The implications of these results for the function of the uvsY protein in T4 DNA replication in vivo are discussed.     

An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.     

Cloning, purification, and properties of Candida albicans thymidylate synthase.

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same.     

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene.

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.     

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes.

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.     

Molecular cloning, sequencing, and mapping of the bacteriophage T2 dam gene.

Bacteriophage T2 codes for a DNA-(adenine-N6)methyltransferase (Dam), which is able to methylate both cytosine- and hydroxymethylcytosine-containing DNAs to a greater extent than the corresponding methyltransferase encoded by bacteriophage T4. We have cloned and sequenced the T2 dam gene and compared it with the T4 dam gene. In the Dam coding region, there are 22 nucleotide differences, 4 of which result in three coding differences (2 are in the same codon). Two of the amino acid alterations are located in a region of homology that is shared by T2 and T4 Dam, Escherichia coli Dam, and the modification enzyme of Streptococcus pneumoniae, all of which methylate the sequence 5' GATC 3'. The T2 dam and T4 dam promoters are not identical and appear to have slightly different efficiencies; when fused to the E. coli lacZ gene, the T4 promoter produces about twofold more beta-galactosidase activity than does the T2 promoter. In our first attempt to isolate T2 dam, a truncated gene was cloned on a 1.67-kilobase XbaI fragment. This construct produces a chimeric protein composed of the first 163 amino acids of T2 Dam followed by 83 amino acids coded by the pUC18 vector. Surprisingly, the chimera has Dam activity, but only on cytosine-containing DNA. Genetic and physical analyses place the T2 dam gene at the same respective map location as the T4 dam gene. However, relative to T4, T2 contains an insertion of 536 base pairs 5' to the dam gene. Southern blot hybridization and computer analysis failed to reveal any homology between this insert and either T4 or E. coli DNA.