Characterization of human cytomegalovirus-induced DNA polymerase and the associated 3′-to−5′, exonuclease

Abstract

A DNA polymerase activity induced by human cytomegalovirus (HCMV) was separated from host cell DNA polymerase and purified by phosphocellulose and DNA-cellulose column chromatography. The DNA polymerase activity was strongly inhibited by phosphonoacetic acid, aphidicolin, araATP, and N-ethylmaleimide, but it was resistant to 2′,3′dideoxyTTP. The sensitivity of HCMV-induced DNA polymerase to these reagents resembles that of host cell DNA polymerase α. However, HCMV-induced DNA polymerase activity was stimulated several fold by 100 m M ammonium sulfate, by which DNA polymerase α activity was strongly inhibited. Furthermore, it was found that a 3′-to−5′ exonuclease activity was tightly associated with the HCMV-induced DNA polymerase. The exonuclease was also stimulated by ammonium sulfate, was inhibited by phosphoacetic acid, and it preferred single-stranded DNA as a substrate. The results suggest that the 3′-to−5′ exonuclease may play a role in proofreading in the polymerization process as an integral part of the HCMV-induced DNA polymerase.