Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.Molecular chaperones regulate many cellular processes such as protein folding and translocation and the assembly and disassembly of multiprotein complexes (reviewed in Ref. 1). Two major Escherichia coli chaperones DnaK and DnaJ were originally identified as genes required for the initiation of bacterial or bacteriophage DNA replication. Mutations in DnaK and DnaJ lead to defects in DNA and RNA synthesis, cell division, and proteolysis (for reviews, see Refs. 2-4). DnaK is a weak ATPase with the ability to bind unfolded polypeptides (5, 6). DnaJ functions as a dimer (7, 8) and is considered a cochaperone, since it dramatically stimulates the ATPase activity of DnaK in the presence of GrpE (6, 9). Together, these proteins facilitate the binding of the replication initiator protein to the origin (ori) and the initiation of DNA replication (Ref. 7; for reviews, see .The families of eukaryotic heat shock protein 70 (Hsp70/ Hsc70) and heat shock protein 40 (Hsp40) have a high degree of homology to DnaK and DnaJ, respectively. Hsp70 and Hsp40 proteins are co-localized to the cytosol and also function in the nucleus (15, 16). As in prokaryotes, the Hsp40 proteins function as co-chaperones of Hsp70, but they also have weak, independent activity (5,7,(17)(18)(19)(20). All of the DnaJ homologues, such as HDJ-1, HDJ-2, and YDJ-1, members of the human and yeast Hsp40 family, have a conserved J domain at the amino terminus. Truncated E. coli or YDJ-1 containing only the J domain is sufficient to modulate the ATPase activity of DnaK/Hsp70 (20, 21). The corresponding J domain of the human Hsp40 proteins is also thought to mediate interactions with Hsp70 and regulate its ATPase activity (for a review, see Ref. 8). Within the J domain, there is a highly conserved HPD tripeptide loop flanked by two ␣-helices, designat...

We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cellfree HPV-11 origin (ori) replication. The E1 protein binds specifically to a wild type but not to a mutated sequence in the ori spanning nucleotide position 1. It also has a relatively strong affinity for nonspecific DNA. A neutralizing antiserum directed against the aminoterminal one-third of the E1 protein totally abolishes initiation and elongation, suggesting that it functions as an initiator and a helicase at the replication fork. An antiserum against the carboxyl-terminal portion of E1 protein abolished replication only when added prior to initiation. Thus this portion of E1 is hidden in the replication complexes. The HPV-11 E2 protein appears not to be essential for elongation, but it must be present in the preinitiation complex for the E1 protein to recruit host DNA replication machinery to the ori. E2 antiserum added after preincubation in the absence of the cell extracts totally abolished replication. An identical conclusion is also reached for the bovine papillomavirus type 1 E2 protein. Finally, the HPV-11 E2C protein lacking the transacting domain of the full-length E2 protein partially inhibits E2-dependent ori replication.The large family of human papillomaviruses (HPVs) 1 cause persistent or recurrent epitheliotropic lesions, some of which can progress to high grade dysplasias or carcinomas (1). Productive infections normally cause exophylic or flat warts in which the viruses have two distinct modes of DNA replication. A low copy number of viral DNA is maintained in basal and parabasal cells that are capable of cell division. Only in a subset of cells undergoing terminal squamous differentiation does vegetative viral DNA amplification take place (2). There is considerable interest in investigating the mechanisms of papillomaviral DNA replication; they may serve as models for host DNA replication as alternatives to SV40 and polyomavirus, and means might be identified to suppress or eradicate persistent infections. However, because of their stringent dependence on squamous epithelial differentiation, these viruses cannot be propagated in cells cultured by conventional means, making it difficult to investigate viral DNA replication.Two assays have been developed to study DNA replication of HPVs and bovine papillomavirus type 1 (BPV-1) which cause fibropapillomas in cattle. One is transient replication of plasmids in mammalian cells cotransfected with expression vectors for viral genes. The other is cell-free replication in the presence of viral proteins purified from insect Sf9 cells infected with recombinant baculoviruses. These assays have demonstrated that replication requires a viral origin of replication (ori), virusencoded E1 and E2 proteins and the host DNA replication enzymes including DNA polymerase ␣/primase, proliferating cell nuclear antigen/DNA polymerase ␦ or ⑀, single-stranded DNA binding protein RPA, and topoisomerases I and II (...

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesintreated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.The cyclopropylpyrroloindole (CPI) 1 drugs are a group of DNA sequence-specific minor groove binders that alkylate the N-3 of adenine at the 3Ј end of the binding sites. CPI drugs are currently in clinical trials for several types of solid tumors (1, 2). The CPI drug, adozelesin, carries a single cyclopropyl group and alkylates a single adenine (3-6). CPI adduct formation on naked DNA is able to block progression of DNA polymerases and helicases (7-9). Previous studies have shown that two different CPI drugs, adozelesin and bizelesin, inhibit both the initiation and elongation stages of cellular and viral DNA replication in cultured cells (10 -12). However, the concentrations of these drugs required to cause S phase arrest are 2-4 orders of magnitude lower than levels of drug required to cause detectable adducts and to block polymerase or helicase progression. These results suggest that inhibition of DNA replication and cell cycle progression in cultured cells upon treatment with CPI drugs occurs via a trans-acting mechanism rather than by directly blocking DNA replication fork progression. The most likely explanation for this trans-inhibition of DNA replication is through cellular DNA damage response pathways or checkpoints.Since both viral, simian virus 40 (SV40), and cellular DNA replication are inhibited at similar CPI levels, it is possible to use the more easily studied viral system to elucidate how CPI treatment results in DNA replication arrest. SV40 DNA replication is the most well studied model for eukaryotic DNA replication. An in vitro system was developed that requires only one viral protein, SV40 large T antigen, an exogenous plasmid DNA template containing the SV40 origin sequence, and primate...

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Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).