DNA polymerases and their role in isolated mouse nuclei

Wood, William McMaster (1974) DNA polymerases and their role in isolated mouse nuclei. PhD thesis, University of Glasgow.

Abstract

1. L929 cultured mouse fibroblasts contain multiple species of DNA polymerase. High-speed supernatant preparations contain one species as revealed by Sephadex G-200 gel filtration. It has a molecular weight in excess of 200,000 daltons and has a preference for denatured DNA as template. An 0.4H KC1 extract of nuclei exhibits multiple peaks of DNA polymerase activity on Sephadex G-200. One of the species (peak I) is similar in size and DNA template preference to the soluble enzyme. Three other species (peaks II, III and IV) are usually found, the major peak (peak III) having a M.W. of approximately 70,000 with the other two species often eluting as shoulders of this central peak. Size considerations suggest a monomer-dimer-tetramer relationship but this has not been firmly established. The smaller nuclear species have a preference for native DNA as template. 2. Previous studies had suggested that deoxyribonucleoside 5'-diphosphates may be nearer, than the triphosphates, to the immediate substrate for the nuclear DNA polymerase. Any conclusions were always complicated by the presence of nucleoside diphosphokinase activity. Fractionation of nuclear extracts shows that for nuclear peak I activity, incorporation of diphosphates, even in the presence of a phosphate donor, is not demonstrable due to the absence of nucleoside diphosphokinase activity. 3. An activity capable of stimulating DNA polymerase by providing RNA primers with 3'OH ends is not detectable in soluble or nuclear fractions of L929 cells, 4. An activity capable of converting soluble DNA polymerase into smaller active fragments typical of the smaller nuclear species is not detectable in supernatant or extracted nucleic However nuclear extracts were not tested as they already contained substantial nuclear species. 5. Centrifugation and agarose gel filtration studies suggested that the soluble enzyme was very large (>0.5 x 10.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: R LP Adams
• Keywords: Biochemistry
• Date of Award: 1974
• Depositing User: Enlighten Team
• Unique ID: glathesis:1974-72100
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 13:00
• Last Modified: 17 May 2019 13:00
• URI: https://theses.gla.ac.uk/id/eprint/72100