Structural studies of other polymerases including Klenow Fragment, T4, T7, and RB69 DNA polymerase have revealed that the distance between the polymerase and exonuclease sites is around 20–30 Å,40−44 significantly closer than in UL30. We performed a series of helicase assays on partially double-stranded substrates that contained either 5′ or 3′ ssDNA overhangs of various lengths (Fig. We speculate that a conformational change of ICP 8 induced by single-stranded DNA caused this effect. Several studies using mutated duplex oligonucleotide allow us to suggest a number of base-specific contacts between OBP and DNA (11, 12). The bands at 90 kDa represent UL5 helicase and are highlighted by an arrow. After 48 h, live cells were stained with PicoGreen, a DNA dye previously shown to label mt DNA nucleoids (3), and MitoTracker Deep Red, which marks the mitochondrial network. The weak binding site for helicase (Box III) was absent.
As shown in Fig.4, a comparison of these two chromatographs revealed a large peak from both columns representing the free DNA and two additional smaller peaks from the column loaded with the cross-linked material. The orientations [(θ, φ), listed below each image] were determined by assuming icosahedral symmetry. 1, ligation of the Uc-containing and Ub-containing termini of these two genomes can potentially generate two additional junction fragments of 2.8 and 4.8 kbp. The ability of UL12 to release well DNA was not dependent on the concentration of UL12, the concentration of DNA in agarose, or the time postinfection at which well DNA was harvested from infected cells (data not shown). Once assembled, however, it appears to be stable and to be able to carry out processive DNA synthesis for an extended period. 2). They also suggest that the UL9 protein undergoes a significant conformational change upon binding Box I.
3C, panel t), respectively. A single-stranded oligonucleotide corresponding to the upper strand of oriS, oriS136, was synthesized. The inability of UL9 to support branch migration is not unexpected given the 5′ overhang of the displaced strand and the 3′-5′ polarity of UL9. At 10 and 12 h (Fig. These results suggest that the target of T157602 is encoded by the viral genome. DNA-binding site sizes of 12–22 nt/ICP8 monomer have been previously determined from electron microscope studies (36) or from indirect measurements based on the concentration of ICP8 required to optimize protein activity or nuclease protection (2, 14, 32, 37). Aliquots were removed after 0, 1, 3, 6, 9, or 15 min, and the RNAs were electrophoresed through 1.3% (wt/vol) agarose-formaldehyde gels.
Differences in sensitivity might also underlie variable accounts of lytic gene expression in latency (e.g. Complementation abilities of zinc finger mutants were determined as described in Materials and Methods. Synthesis of Pt-bis-Nt and related molecules were carried as described elsewhere (Grokhovsky et al., 1992 Grokhovsky, S. (1) In assays containing a large excess of DNA, UL30 (exo+) and UL30 (exo−) accumulate mismatch products at similar rates.6 If the product resulting from incorporation of a wrong dNTP had efficiently translocated to the exonuclease active site, the highly active exonuclease should have significantly decreased the accumulation of the mismatched product. What then is the effect of the additional nucleotides of the single-stranded tail that are protected by OBP from exonuclease I digestion? Latently infected TG were removed immediately, cut into eight equal-size pieces, and cocultivated with 3 × 104 Vero cells in 1.5 ml of complete DMEM per well in 24-well plates. However, direct transmission of cells infected by KSHV cannot be excluded, as this mechanism has been reported in some cases of post-transplant KS (Barozzi et al., 2003; Luppi et al., 2003).
Pull-down assays were performed as described above by adjusting sample volumes to 500 μl with the lysis buffer (100 mM KCl). Each fraction was monitored for protein by 280-nm-absorption readings on a Beckman DU 640 spectrophotometer. To examine the possibility that a single-stranded region might be required to promote the unwinding of box I, a box I duplex was synthesized to which ssDNA strands were attached downstream of the box I sequence (Fig. The proposed helicase for HCMV was first identified as the homologue for HSV-1 UL5. We have employed electrophoretic mobility shift assay (EMSA), fluorescence polarization, and electron microscopic (EM) assays to examine the structure of the ssDNA-protein complexes and the effects of oligomerization on DNA binding kinetics. However, repeated a sequences on the plasmid could also mediate recombination in the absence of HSV-1 infection. Given the variety of contexts in which LANA is transcribed during the virus life cycle, it is clear that LANA homologs also have other important functions during the rhadinovirus life cycle.
The “back face” (opposite face to the side that binds Pol) of a UL42 molecule contains several positively charged residues. These studies predicted an interaction between ICP8 and the helicase-primase heterotrimer mediated by the UL8 protein, which has subsequently been demonstrated (14).