Temperature-sensitive DNA polymerase induced by a bacteriophage T5 mutant: relation between polymerase and exonuclease activities

Abstract

DNA polymerase induced by bacteriophage T5ts53, a mutant with temperature-sensitive polymerase, was purified to .apprx. 95% purity as judged by dodecyl sulfate gel electrophoresis. The 3'' .fwdarw. 5'' exonuclease associated with the polymerase had higher activity than that associated with the parent wild-type enzyme. It was more stable to heat than the polymerase, and it degraded primer-template even in the presence of 4 dNTP''s [deoxynucleoside triphosphates] at higher temperature. The evidence presented shows that the inhibition of DNA synthesis by higher temperature was primarily due to defects in polymerase function rather than overactive exonuclease. The presence of primer-template DNA stabilized the polymerase to heat. Purified ts53 polymerase discriminated against incorporation of BrdUMP [bromodeoxy UMP], especially at higher temperature. This is in agreement with observations made in vivo with ts53-infected bacteria [Escherichia coli].