TY - JOUR
T1 - DNA primase-DNA polymerase α from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates
AU - Yamaguchi, M.
AU - Hendrickson, E. A.
AU - DePamphilis, M. L.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1985
Y1 - 1985
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0021868758&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021868758&partnerID=8YFLogxK
M3 - Article
C2 - 2581949
AN - SCOPUS:0021868758
SN - 0021-9258
VL - 260
SP - 6254
EP - 6263
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -