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. (A) The sequences of the oligonucleotides used to prepare artificial substrates are shown. In an experiment where HisΔOBP and the box I duplex oligonucleotide were mixed at an equimolar ratio a complex, HisΔOBP/box I, eluted at an apparent molecular weight of 92,000. It is noteworthy that bovine serum albumin did not simply prevent irreversible denaturation of HisΔOBP but could be added to a highly diluted sample and restore high affinity binding (results not shown). Panel A.I shows images taken at a low cutoff value also applied in panels B to D. 55 kDa), while AN1 failed to produce either protein (Fig. In solution, the oligonucleotide can form stem and loop structure stabilized by intramolecular pairing of Box I and Box III sequences.
Heparin-Sepharose chromatography resulted in substantial loss of activity but removed several major contaminating polypeptides. The standard deviations are noted in parentheses. In addition, there are strong densities beneath the penton channel in the virion (Fig. Among these, two are likely to be of primary importance: (i) the absence of RL and RS sequences flanking the minimal cleavage/packaging signal may reduce the efficiency of this element, and (ii) the inversion of adjacent L components within concatemers will alter the relative orientations of the packaging signals, resulting in up to half of the replicated DNA being unpackageable and possibly interfering with the encapsidation process itself. This result suggests either that UL12 is not a resolvase or that the exonuclease activity of UL12 masks any cruciform-resolving activity. (A) The reaction mixture contained [α-32P]dGTP 350 fmol of the UL5/52 subassembly and 50 fmol of DNA polymerase-UL42. Seventeen out of 22 (77%) substitutions mediated by the wild-type Pol were transitions, including 15 G:C-to-A:T and 2 A:T-to-G:C changes (Table 2).
Equivalent amounts of UL9 homodimer and Box I substrate were incubated together with increasing amounts of ICP8 in the absence of ATP. To investigate the question of whether the AAV2 Rep helicase domain can inhibit replication of any DNA substrate when binding is facilitated, we used a well-established assay to investigate the replication of HSV-1 and AAV2 DNA (21, 24, 41, 42). The reaction mixture contained 2 fmol of radiolabeled oriS and 0.4 pmol of OBP. At higher acceptor DNA concentrations (>100 μm), the amount of product ceases to increase, as expected, presumably because the concentration of ICP8 becomes limiting. This polypeptide had the same mobility as the VP26-GFP fusion protein observed in capsids. Mutagenic oligonucleotides were synthesized by Operon Technologies (Alameda, Calif.). 2 b), show that equilibrium binding of ICP8 with ssDNA is not simply entropically driven displacement of cations from polynucleotide as reported for simple peptide-DNA complexes (33-35).
In the first, which was designated AUG1→CCC, the 5′ proximal AUG was changed to CCC. This suggests that DNA contamination is infrequent if not absent in single neuron RNA samples prepared using our chosen method. Texas red was excited at 568 nm; FITC was excited at 488 nm. Journal of Biological Chemistry, 279, 29211–29217.[CrossRef], [PubMed]). In control experiments we incubated UL30 with DNAn–1, 5 μM dCTP, and 100 μM dGTP. The mixture was mixed on ice followed by incubation at 37 °C for 10 min. Mouse corneas were scarified with a 26-gauge needle, tear film was blotted, and 2 × 105 PFU of virus in 3 μl of complete DMEM were placed on each cornea as described previously (6).
Experimental infection by MHV-68 has also proven powerful for differentiation of true latent infection from chronic/persistent infection, a distinction that is very difficult to ascertain in natural Human infections by gammaherpesviruses. GST-ICP0(1-109)- and GST-ICP0(106-241)-expressing plasmids were constructed by cloning PCR fragments obtained with the primers 5′-CGCGGATCCCCATGGAGCCCCGCCCCGGA-3′ and 5′-CGGAATTCTACCCCCCGTCCTCTCGAG-3′ for the former and 5′-CGCGGATCCCCGAGGACGGGGGGAGCGACGA-3′ and 5′-CGGAATTCTAGTCGTCCAGGTCGTCGTCAT-3′ for the latter. Cells and viruses. The purified ICP8 migrated as a single polypeptide with a molecular mass of 120 kDa, following SDS/PAGE and silver staining. They also assessed the role of HSV-2 virion host shutoff protein on innate dsRNA antiviral pathways in human vaginal epithelial cells . The constitutive expression of this viral sensor by glial cells and its upregulation following viral challenge provide circumstantial evidence of a role for DAI in glial responses to DNA viral pathogens. Available data do not exclude such a model for HCMV, because the inhibition of DNA synthesis by drug treatment from the time of infection results in the persistence of both circular and nuclease-sensitive linear HCMV genomes (McVoy and Adler, 1994).
We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. The organization of the HSV-1 genome can therefore be delineated asab-UL-b′a′c′-US-ca(1).