Abstract: |
The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase α (pol α)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase δ (pol δ) holoenzyme, which includes pol δ, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol α-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol α-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol α, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol α-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol δ holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed φX174 DNA, the rate of the leading strand synthesis catalyzed by pol δ holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/ min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol δ holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen. |
Keywords: |
dna-binding proteins; nonhuman; dna replication; protein protein interaction; nuclear proteins; double stranded dna; kinetics; rna synthesis; virus large t antigen; simian virus 40; dna, viral; models, genetic; virus replication; cycline; dna replication origin; molecular biology; antigens, polyomavirus transforming; dna denaturation; proliferating cell nuclear antigen; dna-directed dna polymerase; dna directed dna polymerase alpha; dna directed dna polymerase delta; ribonucleotides; dna primase; primer rna; oligoribonucleotides; simiae; dna polymerase iii; rna nucleotidyltransferases; simian virus; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
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