Enzymes of DNA synthesis and degradation in cells infected with herpes simplex virus

Paton, Robert D (1972) Enzymes of DNA synthesis and degradation in cells infected with herpes simplex virus. PhD thesis, University of Glasgow.

Abstract

The main aim of the project was to purify the virus-induced DNA polymerase from herpes simplex virus-infected baby hamster kidney cells grown in culture. If this could not be carried to homogeneity, then the aim would be at lea.st to separate host DNA polymerase and DNase from the enzyme in order to clarify the relationship of the induced DNA polymerase and DNase and eliminate the interfering effects of the other enzymes. In. the course of the present purification studies involving column chromatography on DEAE-cellulose, hydroxylapatite, Sephhadex G-150, and phosphocellulose, and fractionations using sucrose gradient sedimentation and polyacrylamaide gel electrophoresis, evidence was obtained that at least part of the virus-induced DNase activity could be separated from DNA polymerase especially on hydroxylapatite columns. The question arose whether there were one or two new virus-induced DNA exomuclease(s) because, while one of the peaks of DNase from hydroxylapatite was izolymerase-free the other was closely associated with polymerase and remained so throughout several purification steps. The two virus-induced exonuclease peaks wore compared using several criteria including: sedimentation coefficients, substrate specificities, heat stability and rechromatography. The results showed that the enzymes were in the main similar by these criteria although differences detected by the last two criteria may be significant. On the whole these and other characterisation studies coupled with consideration of earlier work suggested that there was probably only one virus-induced DNA exonuclease, and that this was distinct from the DNA polymerase purified protein. Nevertheless, there is also substantial evidence in favour of there being two distinct virus-induced exonucleases, one polymerase-free, the other polymerase-associated. A further possibility that the polymerase-free exonuclease is a breakdown product of a single virus-induced DNA polymerase-DNA exonuclease protein has not been eliminated completely by studies carried out with a protease inhibitor. The final answer rests upon further purification studies. Using hydroxylapatite column chromatography, enzyme purification of some 200-fold was obtained using activated DNA which was a particularly effective primer after this step. Further purification was hampered by instability of the enzyme but studies on protecting agents revealed that storage at -70

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Advisers: J M Morrison; H M Kerr
• Keywords: Biochemistry, Virology
• Date of Award: 1972
• Depositing User: Enlighten Team
• Unique ID: glathesis:1972-72754
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72754