The Highly Processive DNA Polymerase of Bacteriophage T5

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. 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 (1Orr C.W.M. Herriott S.T. Bessman M.J. J. Biol. Chem. 1965; 240: 4652-4658Abstract Full Text PDF PubMed Google Scholar). This activity is essential for phage DNA replication and phage growth (2De Waard A. Paul A.V. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1241-1248Crossref PubMed Scopus (76) Google Scholar, 3Das S.K. Fujimura R.K. J. Biol. Chem. 1980; 255: 7149-7154Abstract Full Text PDF PubMed Google Scholar). The purified enzyme, like many prokaryotic DNA polymerases, has an associated 3′–5′-exonuclease activity that is active on single- and double-stranded DNA (4Das S.K. Fujimura R.K. J. Virol. 1976; 20: 70-77Crossref PubMed Google Scholar, 5Fujimura R.K. Roop B.C. Biochemistry. 1976; 15: 4403-4409Crossref PubMed Scopus (10) Google Scholar). Early studies with the purified enzyme showed that it had two properties not in DNA a processivity of of on DNA that was that with DNA S.K. Fujimura R.K. J. Biol. Chem. Full Text PDF PubMed Google and the to catalyze extensive strand displacement on DNA R.K. Roop B.C. J. Biol. Chem. 1976; Full Text PDF PubMed Google Scholar). of the was to these two properties of T5 DNA polymerase purified from the cloned T5 DNA polymerase gene and thus of The gene for T5 DNA polymerase has been J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). on of it is a of the protein. I of DNA J. Proc. Natl. Acad. Sci. U. S. A. 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The of T5 DNA polymerase to catalyze extensive strand displacement in the of an accessory is also R.K. Roop B.C. J. Biol. Chem. 1976; Full Text PDF PubMed Google Scholar). of the I of DNA catalyze strand displacement but of the enzyme the in the of accessory coli DNA polymerase I not strand displacement the 5′ of the strand is and the T7 DNA not catalyze its exonuclease activity is reduced J. Biol. Chem. Full Text PDF PubMed Google Scholar). DNA polymerase not catalyze strand displacement J. Biol. Chem. Full Text PDF PubMed Google Scholar). DNA T7 and DNA on DNA to the DNA to of the replication the T7 DNA and coli DNA polymerase I catalyze strand displacement in the of a the polymerase a in which it to the strand synthesis. The of in that and A. J. Biol. PubMed Scopus Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Although the gene for T5 DNA polymerase has been cloned Fujimura R.K. 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Full Text PDF PubMed Google Scholar). a with a the activity of T5 DNA polymerase was that for the T7 DNA polymerase and Deletion of 30 residues at the N terminus of T5 DNA polymerase resulted in of the activity of the wild-type T5 DNA polymerase, with of the activity of the wild-type enzyme on Deletion of 24 residues at the C terminus had a effect on its activity on primed Although the activity of T5 DNA polymerase was to that of wild-type T5 DNA polymerase on DNA, it was wild-type T5 DNA polymerase on primed The of activity of T5 DNA polymerase to wild-type T5 DNA polymerase on DNA and primed DNA was and respectively. that the deletions at the C terminus the processivity of to a the at the N terminus. 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We examined the of T5 DNA polymerase to catalyze strand displacement of T5 DNA polymerase the on the the DNA of enzyme the was DNA of strand displacement was by the of in in the of of T5 DNA polymerase to the of T5 DNA polymerase resulted in the of that T5 DNA polymerase has processivity and that it catalyze strand displacement synthesis. the strand displacement by wild-type T5 DNA polymerase is in at the of enzyme, of the had been the of strand displacement with increasing of enzyme, was an increasing of that out strand displacement and the of the T5 DNA polymerase has reduced DNA polymerase activity on primed that it was not to DNA to strand displacement in T5 DNA polymerase, on the out the of strand displacement that with wild-type T5 DNA polymerase not We the exonuclease of wild-type and T5 DNA and The of exonuclease activity of wild-type T5 DNA polymerase was on single- and double-stranded Deletion of the 30 residues at the N terminus reduced the activity on single- and double-stranded DNA by and respectively. deletions at the C terminus of the T5 DNA polymerase dramatically the exonuclease activity. the exonuclease activity of the T5 DNA polymerase was that of the wild-type T5 DNA polymerase by and on single- and double-stranded DNA, respectively. 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Deletion of 30 residues at the N terminus of T5 DNA polymerase had effect on its to carry out strand of 24 residues from the C terminus activity. of a of the unique C terminus of T5 DNA polymerase has a effect at the processivity of the enzyme and its to carry out strand displacement synthesis. Although these two it is also from that the strand displacement by the T5 DNA polymerase with processivity with on The of the of T5 DNA polymerase to catalyze strand displacement in the of proteins is not of the T5 that it an DNA to that in phage replication T7 and The processivity strand displacement by T5 DNA polymerase the for a at the T5 replication it is in for a DNA polymerase to strand displacement from a replication of the it the of of T7 DNA polymerase to strand displacement at M.J. J. Biol. Chem. Full Text PDF PubMed Google and it is that is the for to the growth of T7 S. J. Biol. Chem. 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Full Text PDF PubMed Google and that of T7 DNA is that the of exonuclease in T7 DNA polymerase is an in and that in activity is in by of a replication Deletion of 30 residues at the N terminus of T5 DNA polymerase its double-stranded DNA exonuclease activity by This reduction is not the exonuclease is the of the is that the double-stranded DNA exonuclease activity of T5 DNA polymerase increases by and 24 63 residues from the C respectively. that the C-terminal the of the DNA the exonuclease site, a of activity DNA synthesis. that the of the unique regions at the of T5 DNA of 30 residues from the N terminus of T5 DNA polymerase not effect its polymerase processivity, to strand is that it is in with T5 proteins at the replication of DNA been to be for a the N terminus of DNA polymerase with the for DNA replication J. Biol. PubMed Scopus Google and DNA that processivity DNA polymerase and coli DNA polymerase to the C J. Biol. 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This is in the on the of the polymerase from the it is to that an at the C terminus a of the DNA to the enzyme in a to that of the of a polymerase in the to a the DNA polymerase from the phage a the C terminus with of the DNA Full Text Full Text PDF PubMed Scopus Google Scholar). Deletion of the C-terminal residues of DNA polymerase its processivity and its to DNA PubMed Scopus Google Scholar). for the C of DNA from I and the C terminus with the and the processivity of the We propose that the unique C-terminal of T5 DNA polymerase a the polymerase to DNA, it processivity in the of accessory The of DNA polymerase be of to the for its for out processive DNA synthesis. We for of the with