Stimulation Of Polymerase Activity Research Articles

A novel DNA-binding protein associated with DNA polymerase-alpha in pea stimulates polymerase activity on infrequently primed templates.

A 42 kDa DNA-binding protein is associated with DNA polymerase-alpha-primase in pea (Pisum sativum). In a previous publication it was shown that the protein has strong preference for ds-ss junctions in DNA, including the cohesive termini generated by restriction endonucleases. In this paper it is shown that when the DNA-binding protein is added back to polymerase-primase, the protein stimulates the activity of the polymerase. The stimulation is particularly marked when M13 DNA, primed with a single sequencing primer or primed with oligoribonucleotides by the polymerase's associated primase activity, is used as a template. The stimulation of polymerase activity is not caused by an increase in processivity. These data lead to the suggestion that the 42 kDa DNA-binding protein is a primer-recognition protein.

In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase.

Recent studies have shown that potyvirus VPg/ proteinases and RNA-dependent RNA polymerases are capable of protein-protein interactions in yeast cells. We have extended these studies in vitro. We found that tobacco vein mottling virus (TVMV) VPg is retained on glutathione-Sepharose matrices if co-incubated with a glutathione S-transferase (GST)-NIb fusion protein, but not with GST, which is suggestive of a direct physical interaction between these two proteins. However, a mutation in the VPg (Y1860S) that eliminates virus infectivity and the interaction in yeast cells had little effect on the in vitro interaction. We also found that the TVMV VPg and NIa proteins are capable of stimulating the polymerase activity of the NIb protein. Since this stimulatory activity is retained when the proteinase domain of the NIa is removed, we conclude that the VPg is the moiety responsible for the stimulation of polymerase activity. As with the interaction revealed by co-purification, the Y1860S mutation had little or no effect on the stimulation of polymerase activity. Moreover, the VPg was able to stimulate a mutant NIb with an altered 'GDD' motif. Our studies thus provide two lines of evidence indicative of in vitro interactions between the TVMV VPg and NIb proteins.

The C-Terminal Third of UL42, a HSV-1 DNA Replication Protein, Is Dispensable for Viral Growth

UL42 is the herpes simplex virus type 1 DNA polymerase (Pol) accessory protein and is required for vital DNA replication and growth. Previous results from this laboratory demonstrated that the N-terminal two thirds of the protein contains all of the biochemical activities of the protein which can be measured in vitro. These activities include dsDNA-binding, association with DNA polymerase, and stimulation of polymerase activity. To better understand the functions of UL42 in infected cells, we have isolated and characterized two viral recombinants, UL42lacZ and n338. In the mutant virus UL42lacZ, the UL42 gene was disrupted by insertion of the Escherichia coli lacZ gene, while in the mutant virus n338, a termination codon was introduced after amino acid position 338. Analysis of the mutant phenotypes suggest that (1) the first 338 residues of UL42 retain all the functions necessary for viral DNA replication and growth in lyric infection, (2) localization of UL42 to the cell nucleus is independent of Pol, and (3) localization of ICP8 (ssDNA-binding protein) to prereplication sites is independent of functional UL42.

Transcriptional specificity after mycobacteriophage I3 infection

Transcriptional regulation following mycobacteriophage I3 infection was investigated. For this purpose, RNA polymerase mutants (rifr) of the host bacterium, Mycobacterium smegmatis, were isolated and characterized. Phage growth in rifsand rifr cells in the presence of rifampicin revealed the involvement of host RNA polymerase in phage genome transcription. This was confirmed by studies on in vivo RNA synthesis as well as by direct RNA polymerase assay after phage infection. Significant stimulation in RNA polymerase activity was seen following phage infection. The maximal levels were attained in about 60 min post infection and maintained throughout the phage development period. The stimulation of polymerase activity was most pronounced when the phage DNA was used as the template. RNA polymerases from uninfected and phage-infected M. smegmatis were purified to homogeneity. Enzyme purifn. was accomplished by a rapid procedure utilizing affinity chromatog. on rifampicin-Sepharose columns. Subunit structure anal. of the purified RNA polymerase from uninfected and phage-infected cells showed the presence of \alpha, \beta, \beta' and \sigma subunits similar to the other prokaryotic RNA polymerases. In addn., a polypeptide of 79,000 daltons was assocd. with the enzyme after phage infection. The enzymes differed in their properties with respect to template specificity. Phage I3 DNA was the preferred template for the modified RNA polymerase isolated from infected cells, which may account for the transcriptional switch required for phage development.