The inhibition of DNA nucleotidyltransferase by tissue extracts

Burdon, Margery Grace (1963) The inhibition of DNA nucleotidyltransferase by tissue extracts. PhD thesis, University of Glasgow.

Abstract

The synthesis of deoxyribonucleic acid (DNA) is catalysed by the enzyme DNA nucleotidyltrensferase which requires primer DNA and incorporates monophosphate residues from the deoxyribonucleoside 5'-triphosphates into polydeoxyribonucleo-tides. The transferase from ascites tumour cells is inhibited by extracts of liver and kidney and by calf serum and the present study was undertaken to determine the mode of action of these inhibitory factors. DNA nucleotidyltransferase from calf thymus gland was employed as the test system and the characteristics of this enzyme were first determined. It requires magnesium ions and its activity is enhanced by 2-mercaptoethanol and ethylenediaminetetraacetic acid. The optimal concentrations of primer DNA and deoxyribonucleoside triphosphates have been determined and the time course of incorporation of 32P-TMP has been examined. The addition of salts such as sodium chloride inhibits the reaction. It was confirmed that calf serum and extracts of rat liver and kidney inhibited the reaction. The inhibitory factor in calf serum was dialysable and heat-stable and the inhibition of the transferase could be accounted for by the monovalent cations present in the serum. However, the inhibition of the transferase by extracts of rat liver and kidney was found to be due to a non-dialysable, heat-labile component. Fractionation experiments have eliminated hydrolysis of the deoxyribonucleoside 5'-triphosphates by phosphatase as a mode of action of the inhibitor from rat liver, and the inhibitor has been purified to a point where it exhibits very low levels of hydrolytic activity towards deoxyribonucleoside triphosphates. The influence on DNA nucleotidyltransferase of purified inhibitor preparations was found to be due, at least in part, to the action of a nuclease on the DNA primer. Thus, the deoxyribonuclease (DNase) and inhibitory activities parallelled one another very closely during the purification procedure, and showed a similar sensitivity to heat treatment. Part of the inhibition of the transferase was found to be due to the products of the action of the nuclease on the DNA primer, and not merely to the breakdown of the newly synthesised portions of DNA chains. Relatively high concentrations of pancreatic DNase I were required to inhibit the transferase reaction, and concentrations of DNase I which produced a release of acid-soluble products from DNA similar to that produced by the purified inhibitor caused only slight inhibition. The addition of a small amount of splenic DNase II, which produced only a small amount of acid-soluble products, inhibited the transferase reaction strongly. While the inhibitor preparation caused 35% inhibition of DNA nucleotidyltransferase primed with denatured DNA, 105% stimulation was observed when native DNA was substituted for the denatured material. The nuclease in the purified inhibitor preparation has an optimal pH between 6.5 and 8.5, and a requirement for either manganese or magnesium ions. Calcium ions cannot activate nor can they act synergistically in the presence of magnesium. Any increase in the ionic strength of the medium inhibits the liver nuclease. The nuclease hyarolyses thermally denatured DNA approximately 2.5 times more rapidly than native DNA and exhibits some ribonuclease activity. These properties appear to distinguish this enzyme from any hitherto described nuclease. The activities of various enzymes hydrolysing DNA were determined in subcellular fractions of rat liver. The relative activities of enzymes hydrolysing native and denatured DNA did not differ significantly in the fractions, but the recovery of enzymes hydrolysing native DNA. Was appreciably lower than those hydrolysing native DNA. A comparison of the products of digestion of DNA by the liver nuclease after 1 and 22 hr. indicated an endonucleolytic rather than an exonucleolytic mode of action. Oligo nucleotides Isolated after digestion of DNA for 1 hr. inhibited the transferase reaction, but those obtained after 22 hr. did not. Removal of the terminal phosphate groups from the inhibitory oligonucleotides with alkaline phosphatase did not destroy their activity. A "limit" digest of DNA by the liver nuclease contained deoxyribonucleosides and no other derivatives. A 24 hr. digest also contained a small amount of dinucleotides and the deoxyribonucleoside monophosphates of adenine, guanine, cytosine and thymine, in addition to deoxyinosine, thus indicating the presence of a phosphatase in the nuclease preparation. In the products of a 2 hr. reaction, all four bases seemed to be present at the phosphoryl terminal end of the oligonucleotides. Deoxyinosine also seemed to be present in this position, but no evidence was obtained for its occurrence at non-terminal sites. Deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine and deoxyinosine 5'-monophosphate do not inhibit the transferase reaction.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: R MS Smellie
• Keywords: Biochemistry
• Date of Award: 1963
• Depositing User: Enlighten Team
• Unique ID: glathesis:1963-73881
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73881