Structural studies of UL30 showed that the polymerase and exonuclease sites are 45–60 Å apart.22 Figure shows the overall structure of UL30 in which the D368 residue of the exonuclease active site and the D717 and D888 residues of the palm domain are highlighted. Figure 3D shows that the HSV-1 helicase-primase was unable to bind and unwind the cruciform substrate, suggesting that the helicase-primase is not likely to participate directly in HSV-1 Holliday junction branch migration. The striking observation was made that HisΔOBP was completely displaced from the complex and the previously described ICP 8-T65 complex emerged (Fig. Protein-DNA complexes were isolated by nondenaturing polyacrylamide gelelectrophoresis as described under “Experimental Procedures.” The samples were then subjected to SDS-polyacrylamide gelelectrophoresis in gradient gels followed by Western blot analysis using antiserum against HisΔOBP. Typical AAV replicative intermediates appear at 4.7 kb as AAV monomer duplex form (RF1) and at 9.4 kb as AAV dimer duplex form (RF2), as indicated by the arrows on the right. We next examined the ability of the various truncated UL12 proteins to deplete mt DNA when expressed in the absence of other viral proteins. In these studies, we also used duplexes formed upon annealing of the oligonucleotides S12 and S11 or S10 and S11 (, panel B).
Further insight into the subunit structure of OF-1 was obtained using gel filtration chromatography. Organization of the dsDNA inside the HSV-1 virion. To test for the occurrence of end-to-end joining of input genomes, we analyzed mixed infections with HSV-1 P21 and HSV-1 P31. 3A, lane 6) to completion. These findings suggest that leading and lagging strand synthesis are coupled and rolling circle replication is a processive process. The lack of proofreading, therefore, can lead to an increased frequency of wrongly inserted bases. 8), suggesting that when the CT10 epitope of the UL9 protein is bound to the Box I substrate, it becomes more accessible to the antibody.
Of note, we have observed 5.3% ± 9.2% and 17.3% ± 4.6% of cells (means ± standard deviations) showing numerous nuclear foci when transfected with Rep40/Rep52-LacI (Fig. Reaction conditions were as above. The electrophoretic mobility of the gDNA product was indistinguishable from that formed by the action of RecA (lane 7) and coincident with that of the major product formed by gradual cooling of denatured dsDNA and ssDNA (lane 3). When the VP26-GFP marker was introduced into a virus genome that specified a null mutation in VP5 (KΔ5Z), the fluorescence observed in those infected cells was uniformly distributed throughout the cell (data not shown) and was similar to that observed in Fig. All of the resistant HSV mutants grew to high titers in Vero cells, indicating that the mutations in the resistant isolates did not significantly impair their growth. We have not investigated the effect of DNA sequence which is known to influence the association constants of ssDNA-binding proteins. Vhs degradation of pBK2 mRNA initiates near the 5′ end.
Numbers in brackets show the number of individual cells analyzed. Putative zinc binding residues are highlighted, and mutated residues are underlined. Pt-bis-Nt and Pt∗-bis-Nt contain a glycinated cis-diammino-platinum-group which bridges two netropsin-like fragments. Interestingly, UL42 did not enhance the coordination of the polymerase and exonuclease activities even though UL42 helps tether the polymerase to DNA. Stoichiometry of the OBP·oriS* Complex—OriS* is an efficient activator of ATP hydrolysis catalyzed by OBP (12). Plaques initiated by infectious centers (single latently infected cells) were counted. The virus appears to maintain latency in oral epithelium (Triantos et al., 2004; Pauk et al., 2000), but significant amounts of encapsidated KSHV can be found free of cells in salivary fluid (Vieira et al., 1997; Blackbourn et al., 1998), and are transmissible to cultured cells (Duus et al., 2004; Vieira et al., 1997).
In vitro protein synthesis.Plasmid pT7110 containing ICP0 encoding cDNA (22) was used for in vitro synthesis of [35S]methionine-labeled ICP0 by using a rabbit reticulocyte lysate system (TNT Quick-Coupled Transcription/Translation System; Promega) according to the manufacturer’s recommendations. ICP8-FM was dialyzed in CAPS buffer overnight. Although hydrolysis of the ATP was observed, the UL9 protein was unable to unwind the box I duplex (data not shown). As in all herpesviruses the HCMV helicase–primase is a heterotrimeric complex composed of a helicase subunit (UL105), a primase subunit (UL70) and a linking subunit (UL102). In this study, we have continued our biochemical characterization of the ORF6 protein by examining its ssDNA binding properties. Recombination mediated by the a sequence could be reproduced in a plasmid-based recombination system, which demonstrated that thea sequence is a recombinational hot spot (13, 14). Similar data have been generated for KSHV as well (4, 5).
Nevertheless, the structure of UL42 is very similar to a monomer of the sliding clamp PCNA (39). A third subunit, the 80-kDaUL8 gene product, is an essential component of the helicase-primase complex.