Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit.

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.     

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.     

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.     

Crystal structure of the C-terminal domain of human DNA primase large subunit

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes’ large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.     

The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro.

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.     

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.     

Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation

The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex. The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S. The calculated molecular weight of the reconstituted complex is 312,000. We infer that the stoichiometry of the complex is one of each subunit per complex. The complex has a prolate ellipsoid shape with an axial ratio of approximately 16. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody. While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits). The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact. However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity. The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit. The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase alpha. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wild-type protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.     

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.     

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

The yeast DNA polymerase-primase complex: genes and proteins

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.     

Purification and properties of an accessory protein for DNA polymerase alpha/primase

A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast.     

Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.     

A brain L-type calcium channel alpha 1 subunit gene (CCHL1A2) maps to mouse chromosome 14 and human chromosome 3.

A rat brain cDNA probe was used to localize a gene encoding the alpha 1 subunit of neuronal dihydropyridine-sensitive L-type calcium channels in the mouse and human genomes. Hybridization of the probe to Southern blots made with DNAs from a Chinese hamster x mouse somatic cell hybrid panel indicated that this gene maps to mouse chromosome 14 (Chr 14). Southern blot analysis of an intersubspecies cross demonstrated that the calcium channel alpha 1 subunit gene, termed Cchl1a2, can be positioned 7.5 cM proximal to Np-1. Similarly, segregation among human X rodent somatic cell hybrids indicated that CCHL1A2 maps to human chromosome 3. These assignments are consistent with a region of linkage homology between human chromosome 3p and a proximal region of mouse Chr 14.     

Role of the p68 subunit of human DNA polymerase alpha-primase in simian virus 40 DNA replication

DNA polymerase alpha-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.     

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.