Role of DNA polymerase α and DNA primase in simian virus 40 DNA replication in vitro Journal Article
| Authors: | Murakami, Y.; Wobbe, C. R.; Weissbach, L.; Dean, F. B.; Hurwitz, J. |
| Article Title: | Role of DNA polymerase α and DNA primase in simian virus 40 DNA replication in vitro |
| Abstract: | The role of DNA polymerase α (pol α) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol α and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol α immunoaffinity column resulted in the loss of replication activity. The addition of purified pol α-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol α-primase complex isolated from either mouse cells of calf thymus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol α-primase complex isolated from HeLa cells activated mouse cell extracts. Pol α and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol α or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol α alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associated with pol α, displace the non-template strand, and allow the enzyme to replicate through duplex regions. |
| Keywords: | nonhuman; dna polymerase; dna replication; simian virus 40; cell extract; dna primase; simiae; simian virus; priority journal |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 83 |
| Issue: | 9 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1986-05-01 |
| Start Page: | 2869 |
| End Page: | 2873 |
| Language: | English |
| DOI: | 10.1073/pnas.83.9.2869 |
| PROVIDER: | scopus |
| PMCID: | PMC323408 |
| PUBMED: | 3010320 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 18 August 2021 -- Source: Scopus |
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