The low efficiency of translocation between the polymerase and exonuclease active sites may also reflect the large distance between the two sites. It was shown that the bacteriophage T4 UvsW helicase can bind and unwind cruciform substrates (12), indicating a possible role in branch migration during recombination. In the second experiment we examined the influence of the single-stranded oligonucleotide T65 on the formation of a HisΔOBP-ICP 8 complex. HisΔOBP was cross-linked prior to the incubation with the duplex oligonucleotide PE17/18. DNA samples were fractionated on an agarose gel, blotted and hybridized to a 32P-labeled AAV probe. 2A), consistent with the expectation that M127F produces the UL12M185 protein. Their relative contribution to the overall amplitude of quenching can be determined experimentally.
Theinset below the gel depicts an intensity scan (using NIH Image to create a graphical plot) of the region of the stained gel containing the OF-1-associated peptides. The map is colored radially, using the color scheme shown at the bottom. Since the HSV-1 cleavage/packaging mechanism generates genomes with complementary single nucleotide 3′ overhangs at their opposite ends (3, 28), precise end-to-end joining may be possible only in a head-to-tail orientation (i.e., coupling of a Uc-containing to a Ub-containing terminus), which would produce a junction fragment identical to that formed by circularization. At pH 9 in the presence of magnesium, alkaline nuclease degrades equivalent amounts of agarose-embedded salmon sperm DNA (data not shown) and virion DNA (Fig. In contrast, when the competitor was added late, it failed to inhibit synthesis of double-stranded DNA. Perhaps the HSV-1 Pol has an intrinsic activity to insert incorrect bases and subsequently proofread certain errors, allowing for evolutional events. Finally, when the antibody was added to a UL9 protein-Box I complex, followed by ICP8 and ATP, unwinding was completely inhibited (Fig.
These results indicate that the AAV2 Rep helicase activity in the absence of the Rep DNA-binding and endonuclease activities can inhibit the replication of any DNA template as long as it can bind to it. coli SSB binds oriS*. The addition of helicase-primase to preformed JM resulted in the disappearance of JM and the concomitant appearance of both strand exchange products (gDNA and linear ssDNA) (comparelanes 4 and 6). There was either very little or undetectable cytoplasmic fluorescence, which indicated that VP26 was sequestered in the nucleus with VP5. 2, the viral yields obtained from the resistant viruses derived from HSV-2 were 2 to 4 logs greater than those of wild-type virus in the presence of 50 μM T157602; in addition, no plaques were formed by wild-type viruses at this concentration (data not shown). The binding parameters determined using a 32-mer oligonucleotide can probably be considered a good estimate for those of the reaction with ssDNA. In either case, fragments cleaved from the ends would be too small to be seen on a 1.3% (wt/vol) agarose gel.
(D and E) As per B and C, but restricted to Ntrk1+YFP+ neurons. Accession numbers for each virus, as obtained from the National Center for Biotechnology Information database, are as follows: HSV-1, NP044655; HSV-2, NP044523; human herpesvirus 4 (HHV-4), or Epstein-Barr virus (EBV), P03193; HHV-6, NP042936; HHV-7, AAC40757; varicella-zoster virus (VZV), P09270; murine cytomegalovirus (MCMV), Q69153; herpesvirus saimiri (HVS), P14346; human cytomegalovirus (HCMV), P17149; equine herpesvirus 1 (EHV-1), NP041016; and bovine herpesvirus (BHV), NP045306. Figure 2 Chemical structures of netropsin (Nt) and bis-linked netropsin derivatives: Pt-bis-Nt, Pt∗-bis-Nt, Lys-bis-Nt, and 15Lys-bis-Nt. In support of this idea, the intramolecular translocation of DNA from the exonuclease to the polymerase active site was relatively inefficient and the presence of a DNA in the polymerase active site did not inhibit exonuclease activity. In this instance, ICP8 and the helicase domains of OBP will compete for the same stretch of single-stranded DNA and, as a consequence, the ICP8·OBP complex remains stable. After 4 days, the methylcellulose was removed and monolayers were fixed and stained with 20% methanol-0.1% crystal violet. In fact, the titer in saliva is higher than in PBMCs (LaDuca et al., 1998; Koelle et al., 1997; Pauk et al., 2000), and viral DNA can be detected in saliva in individuals with undetectable PBMC virus (Blackbourn et al., 1998).
The primary antibodies were MAbs 11060 anti-ICP0 (1/10,000), 12CA5 anti-HA-1 (Roche), or M5 anti-FLAG (Sigma) epitopes (1 μg/ml), Do-7 anti-p53 (1/100; Dako), and rabbit polyclonal sera, ML-19 anti-human HDAC4 (a generous gift from Sigma), anti-lamin A/C (Santa Cruz), anti-actin (Sigma), anti-acetylated histone H4 (AcH4; Santa Cruz), and anti-acetylated lysine (1 μg/ml; AcLys; Cell Signaling). The column was equilibrated with 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) buffer, pH 9.0. A 32P-labeled, fully duplex box I sequence was incubated with stoichiometric amounts of UL9 protein and ATP in the presence or absence of ICP8. What follows is a description of each HCMV replication protein and the current status with respect to proposed function. The toposiomerases were of particular importance, since inhibition of their activity abolished lytic KSHV DNA replication altogether.