Deoxyribonucleic Acid Polymerases from Yeast

Abstract

Yeast DNA polymerases A and B were purified 5000 – 10000‐fold by chromatography on DEAE‐cellulose, phosphocellulose, DNA‐agarose and DEAE‐Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the – SH reagents N‐ethylmaleimide and p‐chloromercuri‐benzoate but differ in their sensitivity to cytosine‐arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A‐T)] as template‐primer, polymerase B is most active with poly(dA) · (dT)10. Km values for deoxyribonucleoside triphosphates were determined as 3.7–3.9 μM for enzyme A and 1.8–2.4 μM for enzyme B. During active growth of cells polymerase activity increases about 1.4‐fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme in DNA replication.