Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation Journal Article
| Authors: | Murakami, Y.; Eki, T.; Hurwitz, J. |
| Article Title: | Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation |
| Abstract: | DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase α (pol-α), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase δ, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-α extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-α, conditions under which pol-α was markedly inhibited. These findings suggest that interactions between T antigen, pol-α-primase, and HSSB position the pol-α-primase complex on the lagging-strand template for RNA primer synthesis. |
| Keywords: | dna-binding proteins; nonhuman; dna replication; dna synthesis; rna synthesis; virus large t antigen; simian virus 40; dna, viral; virus replication; cycline; antigens, polyomavirus transforming; in vitro; polyacrylamide gel electrophoresis; dna topoisomerase; dna topoisomerases, type i; elongation factor; virus dna; dna directed dna polymerase alpha; dna template; single stranded dna binding protein; templates, genetic; dna primase; oligoribonucleotide; primer rna; oligoribonucleotides; simiae; dna polymerase ii; rna nucleotidyltransferases; simian virus; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; dot hybridization; dna polymerase α; simian virus 40 large tumor antigen |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 89 |
| Issue: | 3 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1992-02-01 |
| Start Page: | 952 |
| End Page: | 956 |
| Language: | English |
| DOI: | 10.1073/pnas.89.3.952 |
| PUBMED: | 1310541 |
| PROVIDER: | scopus |
| PMCID: | PMC48363 |
| DOI/URL: | |
| Notes: | Source: Scopus |
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