The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems Journal Article

Authors: Eki, T.; Matsumoto, T.; Murakami, Y.; Hurwitz, J.
Article Title: The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems
Abstract: The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol-delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y. S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol-alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol-alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol-alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol-alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta-subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol-delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta-subunit in lieu of T4 gp45 in the elongation of singly primed phi-X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.
Keywords: t-antigen; escherichia-coli; invitro replication; protein complex; cell nuclear antigen; large tumor-antigen; helicase activity; sv40 origin; polymerase-iii holoenzyme; primed dna
Journal Title: Journal of Biological Chemistry
Volume: 267
Issue: 11
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1992-04-15
Start Page: 7284
End Page: 7294
Language: English
ACCESSION: WOS:A1992HN48500017
PUBMED: 1348504
Notes: Article -- Source: Wos
Citation Impact
MSK Authors
  1. Jerard Hurwitz
    206 Hurwitz