ABSTRACT429 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The 429 DNA polymerase was purified from phage-infected cells by using poly (dA) All of the known initiation events of DNA replication start from small RNA or DNA primers with the exception of bacteriophage 429 and adenovirus and their related viruses.These viral DNAs employ a viral-coded terminal protein as a primer for their DNA replication (1-6). The phage 429 genome is a linear double-stranded DNA of about 18,000 base pairs, whose 5' ends are linked to a terminal protein of molecular mass 30 kDa (DNA-protein) (7). DNA sequence analysis has shown that the 429 genome has six-base-pair inverted repeats at the termini (8, 9).Recently, we described a soluble enzyme system for 429 DNA replication that uses exogeneous 429 DNA-protein isolated from phage particles as template (2, 3). The system has facilitated elucidation of the basic mechanism of 429 DNA replication (2-5, 10-14). The first step in the replication of 429 DNA is the formation of a covalent complex between free terminal protein (TP) and dAMP, the first nucleotide of the 5' end of 429 DNA, in the presence of 429 DNAprotein as template. The cell-free extract can catalyze both TP-dAMP complex formation and DNA replication (initiation and elongation). Both activities were copurified through several steps of column chromatography as an apparent replication complex (4). Furthermore, the TP-dAMP complex has been shown to be elongated by the purified replication complex (4), indicating that TP-dAMP indeed functions as a primer for DNA replication.The soluble enzyme system also facilitated identification of the gene products required for DNA replication. Thus far, two gene products have been shown to be necessary for initiation of 429 DNA replication: gene 3 (4, 10, 11) and gene 2 (10-12). Gene 3 is the structural gene for the TP (15). We recently found that phage 429 induces its own DNA polymerase (12). The polymerase activity isolated from cells infected with a gene 2 temperature-sensitive mutant was thermolabile compared with wild-type polymerase. This indicates that gene 2 is the structural gene for the 429 DNA polymerase (12).In this report, we describe the purification of 429 TP and 429 DNA polymerase and show that these two proteins are sufficient to catalyze the initiation of )29 DNA replication. These results provide further insight into protein-primed initiation of linear nonredundant duplex DNA replication.
MATERIALS AND METHODSMaterials. Bacillus subtilis SCR 463 (polA phe hisB trp) was used for preparation of phage-infected cell extracts. Escherichia coli strains RLM866 [C600rj mj thr leu pro (XcI857 ABamHI) supE tonA lac] and RLM 841 [C600rj mrecA-, (XcI'km)/pATY2131 (AmPr PL promoter)] were provided by R. McMacken. Phage 429 wild type, sus2 and sus3 (nonsense mutants in genes...
The purpose of this investigator-blinded, five-treatment, crossover human intraoral study was to evaluate the effects of two experimental dentifrice formulations containing either stannous fluoride (SnF2) or sodium fluoride (NaF) packaged with sodium hexametaphosphate in a dual-phase delivery system on demineralization-remineralization using an in situ model system. The experimental dentifrice formulations’ ability to alter demineralization-remineralization was compared to a series of three controls: SnF2-positive control, NaF-positive control and no-fluoride placebo-negative control. The single-section crown model, developed at the University of Iowa, was used to assess the fluoride efficacy of two experimental products versus the placebo containing no fluoride and positive controls. The results of the current in situ study suggest a clinical level of anticaries activity for the experimental SnF2 and NaF dentifrice formulations that was as good as either of the positive controls, when evaluated using polarized light microscopy. This supports the conclusion that the use of the sodium hexametaphosphate ingredient does not interfere with the normal fluoride activity of these toothpastes. In addition, the experimental SnF2 product was numerically better than both the NaF and placebo controls at preventing demineralization of sound root surfaces.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.