Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Ronald T. Hay, Eric A. Hendrickson, Melvin L. DePamphilis

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40 Scopus citations


Analysis of the nucleotide sequences at the 5′ ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3′-purine-dT-5′ in the DNA template and the secondary site was 3′-purine-dC-5′, with the 5′ end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3′-purine-dT-5′ sites and 20% of the 3′-purine-dC-5′ sites were used. Purine-rich sites, such as PuPuPu and PyPuPu, were excluded. The 5′-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5′ end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (DePamphilis et al., 1980DePamphilis et al., 1983Hay and DePamphilis, 1982Hay & DePamphilis, 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.

Original languageEnglish (US)
Pages (from-to)131-157
Number of pages27
JournalJournal of Molecular Biology
Issue number2
StatePublished - May 15 1984

Bibliographical note

Funding Information:
This work was supported by the Kational Institutes of Health grant CA15579 and by the American Cancer Society grant MV-114. R.T.H. was supported by a Damon Runyon--Walter Winrhell Cancer Fund postdoctoral fellowship. E.A.H. was supported by a National Research Service Award.

Copyright 2014 Elsevier B.V., All rights reserved.


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