Cloning and overexpression of the gene encoding bacteriophage T5 DNA polymerase

Chatterjeea, Deb K.; Fujimurab, Robert K.; Campbella, James H.; Gerarda, Gary F.

doi:10.1016/0378-1119(91)90004-u

Cited by 8 publications

(9 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As expected, the first 10 residues of the cloned gene product (MCNEKLSGRL) were the same as those predicted by Chatterjee et al (17) (Fig. 2A).…”

Section: N Terminus Of Phage T5 Dna Polymerasesupporting

confidence: 74%

“…In 1991, Chatterjee et al (17) corrected the sequence, showing that the original report had a frameshift at the beginning of the gene. The start site in the revised sequence was reported to be the first ATG in the open reading frame, which would result in the production of a 94,000-dalton protein.…”

Section: N Terminus Of Phage T5 Dna Polymerasementioning

confidence: 99%

“…Full-length protein refers to the position of the initiating methionine determined by sequencing the first 10 residues of the DNA polymerase purified from the phage. T5 N⌬30 pol refers to the position of the initiating methionine residue proposed previously (17). B, cloning of the gene for wild-type T5 DNA polymerase.…”

Section: Cloning Strategymentioning

confidence: 99%

“…We initially cloned the gene for T5 DNA polymerase into the vector pRSET-C based on the sequence of the T5 DNA polymerase gene, reported previously (17), to generate the plasmid pRSET-T5. However, our terminal amino acid sequence analysis of the T5 DNA polymerase purified from phage T5-infected cells showed that the start of the gene was 90 bp upstream from that published previously.…”

Section: Cloning Strategymentioning

confidence: 99%

“…Although the gene for T5 DNA polymerase has been cloned (17), the overproduced enzyme was reported to be insoluble, and thus its purification has not been described. In this paper, we report that the actual start site of the gene is 90 bp upstream of that reported previously, resulting in an enzyme of 97,600 daltons.…”

mentioning

confidence: 99%

See 4 more Smart Citations

The Highly Processive DNA Polymerase of Bacteriophage T5

Andraos

Tabor

Richardson

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N-and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3 to 5 double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.Upon infection of Escherichia coli by bacteriophage T5, a new DNA polymerase activity is induced (1). This activity is essential for phage DNA replication and phage growth (2, 3). The purified enzyme, like many prokaryotic DNA polymerases, has an associated 3Ј-5Ј-exonuclease activity that is active on single-and double-stranded DNA (4, 5). Early studies with the purified enzyme showed that it had two properties not found in most DNA polymerases: a processivity of polymerization of nucleotides on single-stranded DNA that was greater than that observed with other DNA polymerases tested (6), and the ability to catalyze extensive strand displacement synthesis on duplex DNA (7). One goal of the present study was to characterize these two interesting properties of T5 DNA polymerase purified from cells expressing the cloned T5 DNA polymerase gene and thus free of any contaminating phage-encoded proteins.The gene for T5 DNA polymerase has been sequenced (8). Based on alignment of homologous regions, it is a member of the pol 1 I family of DNA polymerases (8, 9). The alignment between the large fragment of E. coli DNA polymerase I, T5 DNA polymerase, and T7 DNA polymerase, another phage replicative DNA polymerase, is shown in Fig. 1. T5 DNA polymerase has an extension on each end that is not found in other polymerases from the pol I family. There are an additional 159 residues at the N terminus not present in T7 DNA polymerase (Fig. 1), 130 of which are not p...

show abstract

“…As expected, the first 10 residues of the cloned gene product (MCNEKLSGRL) were the same as those predicted by Chatterjee et al (17) (Fig. 2A).…”

Section: N Terminus Of Phage T5 Dna Polymerasesupporting

confidence: 74%

Section: N Terminus Of Phage T5 Dna Polymerasementioning

confidence: 99%

Section: Cloning Strategymentioning

confidence: 99%

Section: Cloning Strategymentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Highly Processive DNA Polymerase of Bacteriophage T5

Andraos

Tabor

Richardson

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Complete genome sequence of bacteriophage T5

et al. 2005

View full text Add to dashboard Cite

The 121,752-bp genome sequence of bacteriophage T5 was determined; the linear, double-stranded DNA is nicked in one of the strands and has large direct terminal repeats of 10,139 bp (8.3%) at both ends. The genome structure is consistently arranged according to its lytic life cycle. Of the 168 potential open reading frames (ORFs), 61 were annotated; these annotated ORFs are mainly enzymes involved in phage DNA replication, repair, and nucleotide metabolism. At least five endonucleases that believed to help inducing nicks in T5 genomic DNA, and a DNA ligase gene was found to be split into two separate ORFs. Analysis of T5 early promoters suggests a probable motif AAA{3, 4 T}nTTGCTT{17, 18 n}TATAATA{12, 13 W}{10 R} for strong promoters that may strengthen the step modification of host RNA polymerase, and thus control transcription of phage DNA. The distinct protein domain profile and a mosaic genome structure suggest an origin from the common genetic pool.

show abstract

Bacteriophage T5 gene D10 encodes a branch-migration protein

Wong

Sayers

Sanders

2016

Sci Rep

View full text Add to dashboard Cite

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.

show abstract

Cloning and overexpression of the gene encoding bacteriophage T5 DNA polymerase

Cited by 8 publications

References 19 publications

The Highly Processive DNA Polymerase of Bacteriophage T5

The Highly Processive DNA Polymerase of Bacteriophage T5

Complete genome sequence of bacteriophage T5

Bacteriophage T5 gene D10 encodes a branch-migration protein

Contact Info

Product

Resources

About