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...

To understand output control in bacterial chemotaxis, we varied the levels of expression of cellular cheY and cheZ genes and found that the overproduction of the corresponding proteins affected Escherichia coli swimming behavior. In the absence of other signal-transducing gene products, CheY overproduction made free-swimming cells tumble more frequently. A plot of the fraction of the population that are tumbling versus the CheY concentration was hyperbolic, with half of the population tumbling at 30 microM (25,000 copies per cell) CheY monomers in the cytosol. Overproduction of aspartate receptor (Tar) by 30-fold had a negligible effect on CheY-induced tumbling, so Tar does not sequester CheY. CheZ overproduction decreased tumbling in all tumbling mutants except certain flaAII(cheC) mutants. In the absence of other chemotaxis gene products, CheZ overproduction inhibited CheY-induced tumbling. Models for CheY as a tumbling signal and CheZ as a smooth-swimming signal to control flagellar rotation are discussed.

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 (...

Specific apolipoprotein B (apoB) mRNA editing can be performed in vitro on apoB RNA substrates. Native gels and glycerol gradient sedimentation have been used to determine the physical properties of the in vitro editing activity in rat liver cytosolic S100 extracts. ApoB RNA substrates were progressively assembled as 27S complexes for 3 hr with similar kinetics as seen for the accumulation of edited RNA. Assembly was not observed on RNAs from apoB deletion constructs that did not support editing. The 27S complex contained both edited and unedited RNA sequences. Inhibition of 27S complex assembly by vanadyl-ribonucleoside complexes was accompanied by inhibition of editing. Based on these data, we propose that the 27S complex is the in vitro "editosome." A "mooring sequence" model for RNA recognition and editosome assembly has been proposed involving RNA sequences flanking the edited nucleotide.

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