DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates.
DNA primase-DNA polymerase alpha, 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 [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA 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 alpha is modulated by the relative concentrations of ribonucleoside triphosphates.
Yamaguchi
M
M
Hendrickson
E A
EA
DePamphilis
M L
ML
D000818:Animals; D002478:Cells, Cultured; D004247:DNA; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D000882:Haplorhini; D011071:Poly T; D012313:RNA / Q000096:biosynthesis*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology*; D013379:Substrate Specificity
C034477:RNA primers; D012265:Ribonucleotides; D011071:Poly T; D012313:RNA; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II