Purification and characterization of DNA polymerase from the archaebacterium Methanobacterium thermoautotrophicum

• Published in: Biochemistry (Easton) 1986-08, Vol.25 (17), p.4850-4855
• Main Authors: Klimczak, Leszek J, Grummt, Friedrich, Burger, Klaus J
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; DNA polymerase; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Methanobacterium thermoautotrophicum; Transferases
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00365a019

DNA polymerase from the thermophilic archaebacterium Methanobacterium thermoautotrophicum was purified about 16,000-fold by chromatography on heparin-agarose, Blue Sepharose, hydroxylapatite, and phenyl-Sepharose as well as by centrifugation through a glycerol gradient. The enzyme exists in the highly purified preparation as one polypeptide of molecular weight 72K that constitutes its single subunit, as shown by electrophoresis under denaturing conditions, gel filtration, and glycerol gradient sedimentation. With the activity gel technique it is demonstrated that the DNA polymerase is very sensitive to proteolysis and the purified polypeptide could be derived from a larger precursor. The enzyme has a temperature optimum at 65 degree C. Both 3' arrow right 5'- and 5' arrow right 3'-exonuclease activities have been found associated with the DNA polymerase. On the basis of its properties, the enzyme shows certain similarities to prokaryotic DNA polymerases of the type of Escherichia coli DNA polymerase I.