Eucaryotic primase

Abstract

Eucaryotic primase, an enzyme that initiates de novo DNA replication, is tightly associated with polymerase a or yeast DNA polymerase I. It is probably a heterodimer of 5.6 rfI 0.1 S. The enzyme synthesizes oligoribonucleotides of about eight residues which are always initiated with a purine. In vitro the polymerase-primase complex initiates synthesis and pauses at preferred sites on natural single-stranded templates. The relative concentrations of ATP and GTP present in the reaction medium modulate the frequency of site recognition. Primase is strongly ATP-dependent in the presence of single-stranded DNA and of poly(dT). It also synthesizes oligo(rG) in the presence of poly(dC) very efficiently.In the course of the few last years, many laboratories have independently isolated and studied eucaryotic primase. The scope of this review, in bringing available data together, aims towards a better understanding of the enzymatic properties to direct future studies more specifically.This review is limited to nuclear primase. Data on DNA replication in mitochondria [l -6) and on mitochondria1 primase will not be discussed here [7 -91. SITUATION in vivoPriming DNA replication DNA replication has been extensively reviewed [lo-I51 and will not be described here. No known DNA polymerase can initiate the duplication of a genome de novo. The enzyme can only extend replication in a 5' to 3' direction from the free 3'-hydroxyl terminus of a preexisting oligodeoxyribonucleotide or oligoribonucleotide base-paired with the template [12, 161. Replication is continuous on the leading strand (3'-5' orientation) of the template, while it is discontinuous on the opposite strand, the lagging strand (5'-3' orientation). In eucaryotes elongation is accomplished by polymerase a (DNA polymerase 1 in yeast). The DNA fragments synthesized on the lagging strand are limited to 100-200 nucleotides in length 110, 111. Initiation of DNA replication is assumed by an enzyme, primase, which synthesizes oligoribonucleotides complementay to the template. These are called primers or initiator RNA [ll]. Synthesis of initiator RNA in eucaryotes is resistant to high concentrations of a-amanitin, excluding a role for RNA polymerases I1 and 111. A requirement for a primase analogous to the Escherichia coli dnaG gene product was proposed [17]. Once a primer is elongated by DNA polymerase, it is progressively removed to allow the gap separating a nascent DNA fragment from the next one to be filled and the two to be ligated together. Priming nascent DNA chains versus priming DNA replication at the origin of a repliconGenerally, two types of initiation of DNA replication are distinguished: initiation of nascent DNA chains on the lagging strand and initiation of DNA replication on the leading strand at the origin of a replicon. Once DNA is loosened (or dissociated?) from the nucleosome, the unwinding of the double helix and elongation on the leading strand proceeds; initiation of nascent DNA fragments by the polymerase -primase complex on the lagging strand may th...

Single‐stranded DNA binding protein from calf thymus

“…Beside its DNA synthesizing activity, eukaryotic DNA polymerase SI contains an inherent DNA primase activity, which is localized on the two smallest subunits of this enzyme [36]. Somewhat unexpectedly, SSB-covered ssDNA is not an efficient substrate for the primase.…”

DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase

“…activity of DNA-polymerase-a-primase was reported to be extremely sensitive to high salt concentration (Roth, 1987). Despite the high concentration of soluble Mp48 protein, phosphocellulose fractions of p48 lost primase activity after dialysis against the buffer without NaCl (Fig.…”

“…The polymerization products of DNA primase have a characteristic size distribution of 8 -11 nucleotides or multiples thereof (Roth, 1987;Podust et al, 1992;Sheaff and Kuchta, 1993). The RNA primers synthesized by the purified recombinant enzymes were thus analysed by electrophoresis in denaturing gels (Fig.…”

Eukaryotic DNA primase abortive synthesis of oligoadenylates

“…i, INTRODUCTION DNA replication in vivo requires preliminary formation of oligoribonueleotides of about 10 monomers in length, which serve as primers for the synthesis of Okazaki fragments [1-.3], DNA primase, an enzyme responsible for the synthesis of RNA primers was purified from various sources both as separate enzyme and in complex with DNA polymerase ~ [4][5][6][7][8][9], (for earlier references see [10]). This enzyme was shown in vitro to synthesize processively discrete-length oligoribonucleotides of an apparent length of 7-12 monomers and multimeric oligoribonucleotides of a model length of about 10 monomers [4-'/, [11][12][13][14][15][16][17][18][19][20][21].…”