Purification and characterization of RNA polymerase II holoenzyme from Schizosaccharomyces pombe

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart in Saccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified from S. pombe. This stimulation was species-specific, because S. pombe Mediator could not stimulate TFIIH purified from S. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.     

Structure and activity associated with multiple forms of Schizosaccharomyces pombe DNA polymerase delta

DNA polymerase delta (Pol delta) isolated from Schizosaccharomyces pombe (sp) consists of at least four subunits, Pol3, Cdc1, Cdc27, and Cdm1. We have reconstituted the four-subunit complex by simultaneously expressing these polypeptides in baculovirus-infected insect cells. The properties of the purified cloned spPol delta were identical to the native spPol delta isolated from S. pombe cells. In addition, we also isolated a three-subunit complex containing Pol3, Cdc1, and Cdm1. Both three- and four-subunit complexes required replication factor C and proliferating cell nuclear antigen for DNA replication. However, in the presence of low levels of polymerase complexes, the three-subunit complex was less efficient than the four-subunit complex in supporting DNA replication. The inefficient synthesis of DNA by the three-subunit complex can be remedied by the addition of Cdc27, the subunit missing in the three-subunit complex. Gel filtration analysis demonstrated that the three-subunit complex is a monomer of the heterotrimer (Pol3, Cdc1, and Cdm1) and that the four-subunit complex is a dimer of the heterotetramer (Pol3, Cdc1, Cdc27, and Cdm1), similar to the structure of native spPol delta. We have further shown that Cdc1 and Cdc27 interact to form a heterodimeric complex. Gel filtration studies indicate that the structure of this complex is dimeric. These observations suggest that the Cdc27 subunit may play an important role contributing to the dimerization of Pol delta.     

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.     

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.     

The influence of the Cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase delta

Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits, pol 3, Cdc1, Cdc27, and Cdm1. In this report, we examined the role of Cdc27 on the structure and activity of pol delta. We show that the four-subunit complex is monomeric in structure, in contrast to the previous report that it was a dimer (Zuo, S., Bermudez, V., Zhang, G., Kelman, Z., and Hurwitz, J. (2000) J. Biol. Chem. 275, 5153-5162). This discrepancy between the earlier and recent observations was traced to the marked asymmetric shape of Cdc27. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region (amino acids (aa) 1-160) binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restored processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells conferred viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme.     

Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme

Mammalian DNA polymerase delta was originally characterized as a tightly associated heterodimer consisting of the catalytic subunit, p125, and the p50 subunit. Recently, two additional subunits, the third (p68) and fourth subunits (p12), have been identified. The heterotetrameric human pol delta complex was reconstituted by overexpression of the four subunits in Sf9 cells, followed by purification to near-homogeneity using FPLC chromatography. The properties of the four-subunit enzyme were shown to be functionally indistinguishable from those of pol delta isolated from calf thymus. The physicochemical properties of both the reconstituted heterotetramer and the heterodimer of the p125 and p50 subunits were examined by gel filtration and glycerol gradient ultracentrifugation. These studies show quite clearly that the heterodimer and heterotetramer complexes do not behave in solution as dimeric structures. This issue is of significance because several studies of the yeast pol delta complexes have indicated that the third subunit is able to bring about the dimerization of the pol delta complex. The heterodimer is only weakly stimulated by PCNA, whereas the heterotetramer is strongly stimulated to a level with a specific activity comparable to that of the calf thymus enzyme. These results resolve earlier, conflicting reports on the response of the heterodimer to PCNA. Nevertheless, the heterodimer does have some ability to interact functionally with PCNA, consistent with evidence that the p125 subunit itself has an ability to interact with PCNA. The functional interaction of PCNA with the pol delta complex may likely involve multiple contacts.     

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.     

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3′→5′ proofreading activity and its preferred substrates are small gaps with a 5′-phosphate group. Similarly to Polμ, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1–2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polμ, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polμ. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polβ and Polλ, and unlike Polμ, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polβ, Polλ and Polμ.     

Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase

Schizosaccharomyces pombe has an open reading frame, which we named alr1(+), encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1(+) gene in Escherichia coli and purified the gene product (Alr1p), with an M(r) of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K(m) and V(max) values as follows: for L-alanine, 5.0 mM and 670 micromol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 micromol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1(+) gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1(+) gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.     

The N-terminal region of DNA polymerase delta catalytic subunit is necessary for holoenzyme function

Genetic and biochemical studies have shown that DNA polymerase δ (Polδ) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Polδ, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase δ (ΔN Polδ) from calf thymus. The ΔN Polδ was stimulated as the full-length Polδ by PCNA in a RF-C-independent Polδ assay. However, when tested for holoenzyme function in a RF-C-dependent Polδ assay in the presence of RF-C, ATP and replication protein A (RP-A), the ΔN Polδ behaved differently. First, the ΔN Polδ lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing ΔN Polδ was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Polδ. The present study provides the first evidence that the N-terminal part of the large subunit of Polδ is involved in holoenzyme function.     

Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.     

Carboxyl-terminal domain III of the delta' subunit of the DNA polymerase III holoenzyme binds delta

The delta and delta' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2gammadeltadelta'chipsi). The x-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both NH2- and COOH-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. delta and delta' form a 1:1 heterodimer with a dissociation constant (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) determined by surface plasmon resonance was comparable. Domain III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.

Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase alpha-primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase delta assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single-stranded DNA-binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition-based protein switches in which two proteins vie for contact with RPA.     

DNA structure checkpoint pathways in Schizosaccharomyces pombe

The response to DNA damage includes a delay to progression through the cell cycle to aid DNA repair. Incorrectly replicated chromosomes (replication checkpoint) or DNA damage (DNA damage checkpoint) delay the onset of mitosis. These checkpoint pathways detect DNA perturbations and generate a signal. The signal is amplified and transmitted to the cell cycle machinery. Since the checkpoint pathways are essential for genome stability, the related proteins which are found in all eukaryotes (from yeast to mammals) are expected to have similar functions to the yeast progenitors. This review article focuses on the function of checkpoint proteins in the model system Schizosaccharomyces pombe. Checkpoint controls in Saccharomyces cerevisiae and mammalian cells are mentioned briefly to underscore common or diverse features.     

Fidelity of human DNA polymerase eta

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.