Rapid hydrolysis of deoxynucleoside triphosphates accompanies DNA synthesis by T4 DNA polymerase and T4 accessory proteins 44/62 and 45

A. C. Roth, N. G. Nossal, P. T. Englund

Research output: Contribution to journalArticle

Abstract

Bacteriophage T4 gene 44/62 and 45 accessory proteins stimulate DNA synthesis by T4 DNA polymerase. Using a φX174 DNA template primed with a strand of a restriction fragment, these accessory proteins also greatly increase the hydrolysis of dNTP to dNMP which accompanies DNA synthesis by the polymerase. The rate of formation of a deoxynucleoside monophosphate is up to 10 times the rate of its incorporation into DNA. This rapid hydrolysis depends on the combined presence of the polymerase, the accessory proteins, and the template primer. Whth a φX174 template-primer, dCTP is hydrolyzed more efficiently than the other triphosphates; with activated salmon sperm DNA, dATP and dGTP are the most efficiently hydrolyzed. A likely explanation for the deoxynucleoside triphosphate hydrolysis is that the polymerase when detained at hair pin barriers in the template, will alternately incorporate and then, using its 3' exonuclease activity, remove deoxynucleoside monophosphates. The formation of deoxynucleoside monophosphates by this mechanism is very slow in the absence of accessory proteins probably because the polymerase is less processive and frequently dissociates from the DNA when detained at a hairpin barrier. Supporting this explanation is the fact that a primer with a 3' terminus near a stable hairpin barrier will support high levels of deoxynucleoside monophosphate formation but relatively low levels of incorporation. Addition of gene 32 protein virtually eliminates the hairpin barriers, increases the rate of synthesis, and markedly reduces the rate of deoxynucleoside monophosphate formation.

Original languageEnglish (US)
Pages (from-to)1267-1273
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number3
StatePublished - 1982

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Accessories
DNA-Directed DNA Polymerase
Hydrolysis
DNA
spleen exonuclease
Proteins
Genes
Bacteriophage T4
Salmon
Bacteriophages
Spermatozoa
triphosphoric acid

ASJC Scopus subject areas

  • Biochemistry

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Rapid hydrolysis of deoxynucleoside triphosphates accompanies DNA synthesis by T4 DNA polymerase and T4 accessory proteins 44/62 and 45. / Roth, A. C.; Nossal, N. G.; Englund, P. T.

In: Journal of Biological Chemistry, Vol. 257, No. 3, 1982, p. 1267-1273.

Research output: Contribution to journalArticle

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N2 - Bacteriophage T4 gene 44/62 and 45 accessory proteins stimulate DNA synthesis by T4 DNA polymerase. Using a φX174 DNA template primed with a strand of a restriction fragment, these accessory proteins also greatly increase the hydrolysis of dNTP to dNMP which accompanies DNA synthesis by the polymerase. The rate of formation of a deoxynucleoside monophosphate is up to 10 times the rate of its incorporation into DNA. This rapid hydrolysis depends on the combined presence of the polymerase, the accessory proteins, and the template primer. Whth a φX174 template-primer, dCTP is hydrolyzed more efficiently than the other triphosphates; with activated salmon sperm DNA, dATP and dGTP are the most efficiently hydrolyzed. A likely explanation for the deoxynucleoside triphosphate hydrolysis is that the polymerase when detained at hair pin barriers in the template, will alternately incorporate and then, using its 3' exonuclease activity, remove deoxynucleoside monophosphates. The formation of deoxynucleoside monophosphates by this mechanism is very slow in the absence of accessory proteins probably because the polymerase is less processive and frequently dissociates from the DNA when detained at a hairpin barrier. Supporting this explanation is the fact that a primer with a 3' terminus near a stable hairpin barrier will support high levels of deoxynucleoside monophosphate formation but relatively low levels of incorporation. Addition of gene 32 protein virtually eliminates the hairpin barriers, increases the rate of synthesis, and markedly reduces the rate of deoxynucleoside monophosphate formation.

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