DNA primase-DNA polymerase α from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates

M. Yamaguchi, E. A. Hendrickson, M. L. DePamphilis

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

DNA primase-DNA polymerase α, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(PN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by 'capping' with [α-32P]GTP using vaccinia guanylyltransferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase α is modulated by the relative concentrations of ribonucleoside triphosphates.

Original languageEnglish (US)
Pages (from-to)6254-6263
Number of pages10
JournalJournal of Biological Chemistry
Volume260
Issue number10
StatePublished - Jan 1 1985

Fingerprint Dive into the research topics of 'DNA primase-DNA polymerase α from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates'. Together they form a unique fingerprint.

  • Cite this