Characterization of a Processive Form of the Vaccinia Virus DNA Polymerase

Abstract

We have previously shown that the purified, 116-kDa DNA polymerase encoded by vaccinia virus is inherently distributive, synthesizing only a few nucleotides per template binding event under moderate reaction conditions (W. F. McDonald and P. Traktman, J. Biol. Chem. 269, 31190-31197). These properties would be incompatible with efficient DNA replication in vivo and suggest that the polymerase most probably interacts with accessory proteins that stabilize the template/polymerase interaction. Here we show that a highly processive form of the enzyme is indeed present with cytoplasmic lysates prepared from infected cells, and demonstrate that this form of the enzyme is likely to comprise the DNA polymerase in association with an early viral protein with a native molecular weight of approximately 48K.

Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

“…Most importantly, the eluate retrieved from the fUDG-A20-pol extracts was 56-fold more active in the synthesis of total products, and 740-fold more active in the synthesis of full-length RFII product. This eluate was further subjected to a time course assay, to confirm that the rate of RFII formation was comparable to that which we had observed earlier for the processive polymerase activity found in infected cell extracts (20). As shown in Fig.…”

“…For example, defects in the E9 polymerase or the A20 protein compromise processive synthesis on a primed singlestranded template, whereas defects in the D5 NTPase or the B1 protein kinase do not (4,6,20). To determine whether the defect in UDG had an impact on processive DNA synthesis per se, we prepared cytoplasmic extracts from cells infected with wt virus, Dts27, or Dts30 at both the permissive and non-permissive temperatures.…”

“…In this manner, it was determined that processive synthesis requires both the DNA polymerase (E9) and other early viral protein(s) (20), one of which was purified and identified as the product of the A20R gene (5). It is reasonable to hypothesize that UDG might be a second component of the processivity factor.…”

“…The extracts and eluates prepared from cells induced to overexpress various combinations of fUDG, A20, and pol were therefore assayed for their ability to direct processive synthesis on the same singly primed M13 template that we have used previously to study purified polymerase, cytoplasmic extracts, and enriched preparations of the processive polymerase (4,5,20). These data are shown in Fig.…”

“…This pol protein was purified by conventional chromatography (14); its enzymatic activity is highly distributive under the assay conditions used here (20). The results of such an experiment are shown in Fig.…”