Purification and Partial Characterisation of Rat‐Liver Nuclear DNA Polymerase

Abstract

A method is described for the preparation of DNA polymerase purified about 800‐fold from rat liver nuclei. The yield of enzyme is about 140–200 μg from 200 g liver. Sodium dodecylsulphate‐polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29000 ± 3%. Sephadex G‐100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42000. The enzyme prefers double‐stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside‐5′‐triphosphate in the assay. The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it.

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

“…Although molecular-weight estimations of DNA polymerase II agree closely with those obtained for similar enzymes derived from rat liver (Haines et al, 1972), KB cells (Sedwick et al, 1972;Wang et al, 1974) and calf thymus (Chang, 1973), those of DNA polymerase I components are different from published values from other systems (Craig & Keir, 1974). The detection of polymerase IA and IB (mol.wts.…”

High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea)

“…Electrophoresis at pH 8.3 and 9.3 resulted in aggregation or precipitation of the enzyme between the running and spacer gel. This aggregation behavior is identical with that observed for mammalian 6-8 S DNA polymerases (Haines et al, 1972;Brun et al, 1974). Upon further purification of the enzyme by stepwise elution on DNA-cellulose, a single major stainable band that was obtained in 7% gels (Figure 3) separated in dodecyl sulfate gels into two polypeptide bands with estimated molecular weights of 70 000 and 34 000 (Figure 4).…”

Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight

“…Nucleoside diphosphokinase (ATP-nucleoside diphosphate phosphotransferase, EC 2.7.4.6) may also be an integral part of pol I and pol II of Esch. coli and pol I of M. luteus (Miller & Wells, 1971) and has also been reported in preparations from KB cells (Sedwick et al, 1972), rat liver Ruclei (Haines et al, 1972), Landschutz ascites-tumour cells (Shepherd & Keir, 1966) and T. pyriformis (Crerar & Pearlman, 1974). Its occurrence may be widespread.…”