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We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase . Temperature-sensitive pol5-1ϳ-3 mutants did not arrest at G 2 ͞M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of ⌬pol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units. DNA polymerases (Pols) play important cellular roles in DNA replication, repair, and recombination. Several distinct polymerases have been identified, purified, and characterized from prokaryotic and eukaryotic cells, and these enzymes are classified into several distinct types (1-3). The best characterized eukaryotic polymerases are polymerases (Pols) ␣, ␤, ␦, , and ␥. Recent studies also identified prokaryotic and eukaryotic polymerases that perform translesion synthesis in the presence of DNA damage (3).In Saccharomyces cerevisiae, Pol ␣, ␦, and are required for chromosomal DNA replication (4), and Pol is required for both chromosomal DNA replication and sister chromatid cohesion (5). Pol IV (␤), Rev3 (Pol ), and Rad30 (Pol ) participate in DNA repair and͞or recombination (6), and Pol␥ is the mitochondria DNA (4). The complete sequence of the yeast genome identified an ORF [YEL055C (POL5)] that encodes a polypeptide resembling B-type Pols. This study characterizes the ORF in YEL055C (POL5) and shows that it encodes the fifth essential Pol in S. cerevisiae. To investigate its function in vivo, several conditionally lethal mutants and a polymerase active-site mutant of POL5 were generated. These mutants were not defective in chromosomal DNA replication, and the polymerase active-site mutant gene fully complemented the lethality of a ⌬pol5 mutation. These results suggest that Pol5p plays an essential role in a cellular function other than chromosomal DNA replication.The nucleolus is the subnuclear compartment in which most steps in the production of ribosomes occur, including synthesis of prerRNAs, pre-RNA processing and modification, and ribosome assembly. The nucleolus also plays roles in the synthesis of ribonucleoprotein particles and pre-tRNA processing (7). Subcellular localization studies showed that Pol5p exclusively colocalizes with Nop1p, which is a component of the C ϩ D small nucleolar ribonucleoprotein-binding protein complex (8), in the nucleolus. Therefore, it is possible that Pol5p may perform an essential function in the nucleolus. This possibility is consistent with the phenotype of pol5-3 ...

DNA polymerases ␦ and ⑀ (pol ␦ and ⑀) 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 ⑀, 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 ⑀ was 0.47 ؋ 10 ؊5for G:G mismatches, 0.15 ؋ 10 ؊5 for T:G mismatches, and less than 0.01 ؋ 10 ؊5 for A:G mismatches. The accuracy for A opposite G was not altered in the exonucleasedeficient pol2-4 pol ⑀; however, G:G and T:G misincorporation rates increased 40-and 73-fold, respectively. The pol2C1089Y pol ⑀ 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 ⑀ is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.The yeast Saccharomyces cerevisiae has three DNA polymerases, (pol ␣, ␦, and ⑀), which are required for cell growth, chromosomal DNA replication (1), and DNA double-strand break repair (2). pol ␣ has four subunits (Pol1 (Cdc17), Pol10, Pri1, and Pri2) and is involved primarily in the initiation of DNA replication and priming of Okazaki fragments. pol ␦ and pol ⑀ are required during synthesis of the leading and lagging DNA strands at the replication fork; they bind at/or near replication origins and move along DNA with the replication fork (3, 4). The precise roles of pol ␦ and pol ⑀ during leading and lagging strand DNA synthesis have not yet been defined; however, genetic and biochemical evidence suggest that lagging strand DNA synthesis is carried out by pol ␣ and pol ␦ (5, 6).pol ␦ of S. cerevisiae has three subunits (Pol3 (Cdc2), Hys2 (Pol31) (7,8), and Pol32 (8)), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively (9). S. pombe pol ␦ has one additional subunit, Cmt1 (9). Purified yeast pol ␦ requires accessory factors including PCNA 1 and RFC to catalyze processive DNA synthesis (8). pol ␦ possesses a 3Ј-5Ј exonuclease activity, which performs proofreading/editing during DNA synthesis (10, 11).The S. cerevisiae pol ⑀ is also a multi-subunit protein complex that includes Pol2, Dpb2, Dpb3, and Dpb4 and like pol ␦ has a 3Ј-5Ј exonuclease activity (1, 12). pol ⑀ is a highly processive enzyme (12)(13)(14). Although pol ⑀ requires PCNA and RFC complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA (13), these cofactors may not be required for processive DNA synthesis in vivo. Pol2 is the catalytic subunit of pol ⑀ and is encoded by the POL2 gene (15), which is essential for cell growth and re...

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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.

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To clarify the functions of the Arabidopsis thaliana REV1 (AtREV1) protein, we expressed it in Escherichia coli and purified it to near homogeneity. The deoxynucleotidyl transferase activity of the recombinant AtREV1 was examined in vitro using a primer extension assay. The recombinant AtREV1 transferred one or two nucleotides to the primer end. It efficiently inserted dCMP regardless of the opposite base. AtREV1 also inserted a dCMP opposite an apurinic/apyrimidinic site, which is physiologically generated or induced by various DNA-damaging agents. In contrast, AtREV1 had no insertion activities against UV-inducible DNA lesions as reported in yeast or mammalian system. Although the substrate specificity of AtREV1 was rather narrow in the presence of magnesium ion, it widened in the presence of manganese ion. These results suggest that AtREV1 serves as a deoxycytidyl transferase in plant cells.

We found that a ciliated protozoan, Paramecium, swam perpendicular to a static (DC) magnetic field (0.68 T). The swimming orientation was similar even when the ionic current through the cell membrane disappeared after saponin treatment. To determine the diamagnetic anisotropy of intracellular organs, macronuclei, cilia, and secretory vesicles, trichocysts, were selectively isolated. Both cilia and trichocysts tended to align their long axis parallel to the magnetic field (0.78 T). Paramecium mutants that lack trichocysts also swam perpendicular to the magnetic field, although the proportion fraction was smaller than the normal population. Since large numbers of cilia and trichocysts are arranged at right angles to the long axis of the cell, the diamagnetic anisotropies of cilia and trichocysts cause the long axis of the cell to align perpendicular to the magnetic field. In contrast to the DC magnetic field, an alternative (AC) magnetic field (60 Hz, 0.65 T) had almost no effect on the swimming orientation of Paramecium.