Studies of T4 Polynucleotide 5'kinase 3'phosphatase
Soltis, Daniel Andrew
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https://hdl.handle.net/2142/70175
Description
Title
Studies of T4 Polynucleotide 5'kinase 3'phosphatase
Author(s)
Soltis, Daniel Andrew
Issue Date
1981
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Bacteriophage T4 polynucleotide 5'kinase 3'phosphatase catalyzes two different reactions. Although the biochemical properties of both activities have been relatively well characterized, the role of this enzyme in T4 infection is not understood. By determining the physiological function of this enzyme, a rationale may be found for having both activities on the same polypeptide chain. In addition, little is known concerning the location of the active sites on the polypeptide chain or if the two activities share a single active site.
By purifying three mutants of polynucleotide kinase phosphatase and comparing their physical and enzymological properties to those of the wild type enzyme, results were obtained which imply that the two activities are catalyzed from independent sites. Using these purified mutant enzymes, a model for the function of polynucleotide kinase phosphatase in T4 infection was tested. It was proposed that this enzyme may catalyze the transfer of a phosphate from the 3'side to the 5'side of a nick in DNA as part of some sort of repair mechanism. Although it was demonstrated that polynucleotide kinase phosphatase can transfer phosphates at nicks in DNA, the transfer occurred through two independent reactions which could be catalyzed by two different proteins and thus provided no evidence that rationalizes the presence of the two activities on the same polypeptide chain. These results also suggest that the model proposed for the physiological function of polynucleotide kinase phophatase is incorrect.
The relationship between the active sites of the two activities of polynucleotide kinase phosphatase was examined by treating the enzyme with two chemical modification reagents and three proteases. In every case, conditions were found where one of the two activities could be eliminated without significantly reducing the other activity. Taken together, these results indicate that the two activities have different active sites that are located in independent domains on the polypeptide chain. Based on the specificity of the proteases, the 5'kinase active site is located in the amino-terminal domain and the 3'phosphatase active site in the carboxy-terminal domain.
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