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PubMed1991

Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate.

9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.

Animals↗
PubMed1989

Evidence for the presence of DNA primase in mitochondria of Saccharomyces cerevisiae.

Chromatography of a DNA polymerase preparation from mitochondria of Saccharomyces cerevisiae on DNA-cellulose column, using Tris-HCl (pH 7.5) buffer containing 0.6 M NaCl as eluent, was found to yield a fraction exhibiting DNA primase-like activity free of DNA polymerase. This fraction could support the synthesis of 12-15 residue-long oligoribonucleotides on single-stranded natural or synthetic DNA templates. The oligoribonucleotides could be further elongated by incorporation of deoxyribonucleotides in the presence of Klenow fragment.

Chemical Fractionation↗
PubMed1989

DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization.

Highly purified DNA polymerase alpha-DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti-human DNA polymerase alpha monoclonal antibody SJK 287-38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA-polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.

Chromatography, Affinity↗
PubMed2004

Human DNA primase uses Watson-Crick hydrogen bonds to distinguish between correct and incorrect nucleoside triphosphates.

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha further elongates. Primase readily misincorporates the natural NTPs and will generate a wide variety of mismatches. In contrast, primase exhibited a remarkable resistance to polymerizing NTPs containing unnatural bases. This included bases whose shape was almost identical to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base [e.g., 5- and 6-(trifluoromethyl)benzimidazole], bases much more hydrophobic than a natural base [e.g., 4- and 7-(trifluoromethyl)benzimidazole], bases of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-D-guanine), and bases capable of forming only one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). Primase only polymerized NTP analogues containing bases capable of forming hydrogen bonds between the equivalent of both N-1 and the exocyclic group at C-6 of a purine NTP (2-fluoroadenine, 2-chloroadenine, 3-deazaadenine, and hypoxanthine) and N-3 and the exocyclic group at C-4 of a pyrimidine. These data indicate that human primase requires the formation of Watson-Crick hydrogen bonds in order to polymerize a NTP, a situation very different than what is observed with some DNA polymerases. The implications of these results with respect to current theories of how polymerases discriminate between right and wrong (d)NTPs are discussed.

Base Sequence↗
PubMed1999

Cloning, expression, and purification of Bacillus stearothermophilus DNA primase and crystallization of the zinc-binding domain.

The dnaG gene encoding DNA primase has been isolated from chromosomal DNA of Bacillus stearothermophilus and its entire nucleotide sequence determined. The deduced amino acid sequence comprised 597 amino acid residues and the molecular mass was calculated to be 67068 Da. B. stearothermophilus primase was overexpressed in Escherichia coli and purified to homogeneity. The N-terminal 12 kDa zinc-binding domain has been crystallized. The crystals are of the monoclinic space group P21 with cell dimensions a=36 A, b=59 A, c=46 A, beta=91.8 degrees and diffract to 1.7 A resolution.

Amino Acid Sequence↗
PubMed1986

Preferential binding of DNA primase to the nuclear matrix in HeLa cells.

Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.

Adenosine Triphosphate↗
PubMed1982

Overlapping genes at the DNA primase locus of the large plasmid ColI.

The sog gene of the large plasmid ColIdrd-1 has previously been shown to encode a DNA primase and a smaller antigenically related polypeptide. Genesis of these two products has been examined using Sog+ recombinant plasmids. Effects of amber mutations, isolated after in vitro mutagenesis, and deletions into or within sog suggest that the smaller polypeptide is a separate translation product which is encoded by DNA specifying the C-terminal region of the larger protein. Under control of the lac promotor, synthesis of both polypeptides is reduced when transcription is repressed. These findings imply that transcription of sog yields a single transcript which is translated from two initiation sites.

Acetyltransferases↗
PubMed1987

The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae.

The PRI1 gene of Saccharomyces cerevisiae encodes for the p48 polypeptide of DNA primase. We have determined the nucleotide sequence of a 1,965 bp DNA fragment containing the PRI1 locus. The entire coding sequence of the gene lies within an open reading frame, and there are 409 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame. The 5' and 3' end-points of PRI1 mRNA have been determined by S1 mapping and primer extension analysis. The primary structure and the codon usage of PRI1 suggest that this essential gene is poorly expressed in yeast cells.

Amino Acid Sequence↗
PubMed1999

Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology.

Comparison of the amino acid sequences of eucaryotic DNA primase and the family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies as well as provide insights into the mechanism of primase, we generated three mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases. These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.

Amino Acid Sequence↗
PubMed1999

Cloning and characterisation of the gene for the large subunit of the DNA primase from Drosophila melanogaster.

We have cloned the gene for the large subunit of the DNA primase from Drosophila melanogaster, and mapped it to position 77b on chromosome 3. The central region of the protein shows high similarity with homologues from other species, but the N- and C-termini diverge. The protein is enriched in replicating tissues, and consistent with this the region upstream of the gene contains close matches to the sites of two transcription factors - Dref and E2f - which have been implicated in controlling proliferation-associated genes.

Amino Acid Sequence↗
PubMed2002

The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting.

The p58 subunit of human DNA primase contains a region, M288-K344, that is homologous to part of the 8 kDa domain of DNA polymerase beta. Since regions of a protein that are highly conserved evolutionarily often play important catalytic functions, we examined the effects of mutating this region of the p58 subunit on primase activity. Deleting M288-L313 of the p58 subunit results in a protein that binds to the primase p49 subunit but cannot support primer synthesis on any template when assays only contain Mg(2+) as the divalent metal. Including Mn(2+), a metal that stimulates initiation of primer synthesis, in the assays now allows the enzyme to synthesize primers at a rate only moderately lower than that of the wild-type enzyme on templates consisting solely of deoxycytidylates. While the enzyme is active under these conditions, it has lost the ability to synthesize primers of defined length (i.e., count). Alanine scanning mutagenesis of charged residues in this region revealed three amino acids, R302, R306, and K314, that play important roles in both primer initiation and translocation. Conversion of these residues to alanine interfered with initiation and significantly decreased the processivity of primase. Together, these studies indicate that this "pol beta-like" region of p58 is important for three distinct aspects of primer synthesis:; initiation, translocation, and counting. The implications of these results with respect to the biological role of the p58 subunit and the mechanism of primer synthesis are discussed.

Amino Acid Sequence↗
PubMed1982

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Plasmid ColIb-P9 of the I alpha incompatibility group is known to encode a DNA primase that acts in the conjugal transfer of the plasmid and can substitute for mutant dnaG gene product in vegetative replication of the Escherichia coli chromosome. The relevant genetic determinant (sog) has previously been cloned into a small multicopy vector plasmid. Prototype IncB plasmid R16 also suppresses host dnaG mutations. The equivalent gene(s) (pri) of R16 was cloned into plasmid pBR325 and shown by filter hybridization studies to be different from the ColI primase gene(s). The cloned fragment carrying the pri determinant encoded an enzyme which could initiate DNA synthesis in vitro on single-stranded phage M13 DNA template, but which was antigenically distinct from ColI primase. We used the cloned primase genes as probes in colony hybridization screening of strains carrying plasmids of the IncI complex and IncB group. Plasmids R64, R144 (I alpha), R621a (I gamma), RIP72, and R864a (B) contained nucleotide sequences homologous with the cloned ColI sog genes. Plasmids R805 (I xi), R724, (I delta), TP125, and PLG101 (B) showed sequence homology with the R16 pri determinant.

Chromosomes, Bacterial↗
PubMed1990

Expression of the yeast DNA primase gene, PRI1, is regulated within the mitotic cell cycle and in meiosis.

Mitotic cultures synchronised either by a feed-starve protocol or by elutriation have been used to show that the Saccharomyces cerevisiae DNA primase I gene is periodically expressed in the cell cycle. The transcript increases many-fold in late G1 and reaches a peak at the same time as four other genes essential for DNA synthesis, CDC8, CDC9, CDC21 and POL1. The primase I transcript is also regulated in meiosis, reaching maximal levels during premeiotic DNA synthesis.

DNA Primase↗
PubMed2000

Nucleotide sequence of a 7-kb fragment of pACM1 encoding an IncM DNA primase and other putative proteins associated with conjugation.

A 7-kb fragment of pACM1 (fragment 90¿91) containing one or more kor (kill-override) loci was sequenced, and 28 open reading frames (ORFs; >/=50 codons) were identified. The nucleotide sequence has no significant homologs in the GenBank database except for a 1.3-kb region 98.6% identical to the iml (insensitivity to phage PhiM-mediated lysis) determinant fragment of IncM plasmid R446. Deduced amino acid sequences for several ORFs are homologous to those of known proteins, including the Sog DNA primases of IncI1 plasmids R64 and ColIb-P9 and the TraL, TraM, and TraN products of ColIb-P9. Two protein products of the putative primase ORF (ORF 1, 1100 amino acids) were detected by SDS-PAGE. The 158- and 107-kDa proteins were designated PriL and PriS, respectively. PriS is apparently produced by an in-frame reinitiation of the ORF 1 transcript at a second start codon located between a Sau96I site and a PstI site. The motif EGYATA, conserved among primases and associated with primase function, occurs in the first one-third of the deduced amino acid sequence of PriL and is not included in PriS. Partial suppression of the temperature-sensitive dnaG3 mutation in BW86 was demonstrated by recombinants that overexpressed both PriL and PriS, but not by constructs overexpressing only PriS. Therefore, primase function can be assigned to PriL. Fragment 90/91 represents a portion of the IncM tra region, which has not previously been examined in detail.

Amino Acid Sequence↗
PubMed1999

Human DNA primase: anion inhibition, manganese stimulation, and their effects on in vitro start-site selection.

We examined the effects of Mn(2+) on eukaryotic DNA primase both in the presence and absence of 5 mM Mg(2+). In the absence of Mg(2+), Mn(2+)-supported primase activity to a level 4-fold greater than that obtained with Mg(2+) alone, and adding low levels of Mn(2+) (100 microM) to assays containing 5 mM Mg(2+) greatly stimulated primase. Increased activity was primarily due to more efficient utilization of NTPs, as reflected in a lower K(M) for NTPs. Under conditions of saturating NTPs, Mn(2+) had minimal effects on both the rate of initiation (i.e., dinucleotide synthesis) and processivity. The effects of Mn(2+) involve multiple metal binding sites on primase and may involve both the catalytic p49 subunit as well as the p58 subunit. Physiological levels of salt can inhibit primase activity due to the presence of an anion binding site and low levels of Mn(2+) significantly decreased this salt sensitivity. The implications of these results with respect to the biological role of primase are discussed.

Anions↗
PubMed1985

Isolation and characterization of a DNA primase from human mitochondria.

A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.

DNA Primase↗
PubMed1984

Plasmid RP4 encodes two forms of a DNA primase.

The pri gene locus of the conjugative broad host range plasmid RP4 maps between coordinates 40.3 and 43.5 and encodes two antigenically related forms of a DNA primase with a molecular mass of 118 and 80 kDa (kilodalton). Genesis of these two products has been examined using Pri+-recombinant plasmids. As shown by deletion analysis, the primase polypeptides are tow separate translation products which arise from an in-phase overlapping gene arrangement. It is suggested that transcription of a set of RP4 genes including the pri gene starts at a promoter site within the Tra1 region. In vivo, RP4 mutant primase can apparently substitute for Escherichia coli primase as demonstrated by measuring suppression of the dnaG3 (ts) mutant.

Bacterial Proteins↗