In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl 2 or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of ϳ48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent ϳ45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken Vaccinia virus exhibits a high degree of genetic and physical autonomy from the host cell, possessing a genome that encodes more than 200 genes and directing a replicative cycle in which DNA replication, gene expression, and morphogenesis take place solely within the cytoplasm of the infected cell. It is therefore likely that the trans-acting functions required for DNA replication are virally encoded, and several laboratories have engaged in studies aimed at identifying and characterizing these functions. The catalytic DNA polymerase of vaccinia virus is encoded by the E9 gene (6,20,31). The 116-kDa enzyme possesses both polymerase and proofreading exonuclease activity and retains many of the conserved domains that are found within the replicative polymerases of mammalian and yeast cells, as well as those encoded by other large DNA viruses. In addition, the vaccinia virus enzyme, like the catalytic subunit of these other replicative polymerases, is inherently distributive. Our studies on the purified E9 protein indicate that in the presence of 40 mM NaCl or 8 mM MgCl 2 , the enzyme catalyzes the addition of Ͻ10 nucleotides (nt) per primer-template binding event (21). Such distributive behavior would be incompatible with efficient replication of a large genome in vivo, and it is therefore no surprise that a highly processive form of the vaccinia virus enzyme is found in unfractionated cytoplasmic extracts of infected cells (19). This processive enzyme is capable of catalyzing the addition of Ͼ7,000 nt per primer-template binding event.This dichotomy between the dis...

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.

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Grampositive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli ␤ clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A., and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus ␤ clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80 -120 nucleotides per s. Both E. coli ␤ and S. aureus ␤ converted PolC to a highly processive polymerase, but surprisingly, ␤ also increased the intrinsic rate of DNA synthesis to 240 -580 nucleotides per s. This finding expands the scope of ␤ function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.Numerous proteins cooperate to perform the complicated task of replicating duplex DNA. At the heart of this process lies the replicative DNA polymerase machinery. The chromosomal replicase of Escherichia coli is a large particle, DNA polymerase III holoenzyme, which consists of 10 different proteins in stoichiometries that range from one to four (reviewed in Refs. 1-3). Within the holoenzyme are two copies of the three subunit core polymerase (␣, DNA polymerase (4); ⑀, proofreading 3Ј-5Ј-exonuclease (5); and ) (6 -8). The ␤ subunit of DNA polymerase III holoenzyme is a protein ring that encircles DNA and slides along the duplex (9, 10). The ␤ ring binds the ␣ subunit of core and tethers the holoenzyme to DNA for high processivity in DNA synthesis (9). The five subunits that comprise ␥ complex (␥␦␦Ј) act as a clamp loader to open and close the ␤ ring around DNA in an ATP-driven reaction (11,12).The main bacterial system for study of cellular DNA replication is the Gram-negative E. coli. However, there is a great deal of diversity in the eubacterial kingdom. Perusal of bacterial genome data bases reveals homologs to the E. coli ␤ clamp, ␣ subunit of core polymerase, and at least two (␥ and ␦Ј) of the five subunits of ␥ complex, suggesting that the overall strategy of using a clamp and clamp loader generalizes among this diverse group of organisms (eukaryotes also use a clamp loader replication factor C) and clamp (proliferating cell nuclear antigen)) (13,14).The evolutionary split between Gram-positive and Gramnegative bacteria occurred over 1 billion years ago, and the sequences of the replicative polymerase and ␤ clamp have diverged considerably. Purification of the replicative DNA polymerase, encoded by polC, from a variety of Gram-positive cells yields only a single polypeptide, in contrast to the...

Vaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively, encode type I DNA topoisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT.