The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha

Summary The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 by intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.     

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 (α) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase α. 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.     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

A method to select for mutator DNA polymerase deltas in Saccharomyces cerevisiae.

Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

Cloning and Characterization of the Human Mitochondrial DNA Polymerase, DNA Polymerase γ

The nuclear-encoded DNA polymerase γ (DNA POLγ) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POLγ and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POLγ gene fromDrosophila melanogasterand a partial cDNA for DNA POLγ fromGallus gallushave been cloned. The predicted human DNA POLγ polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those ofSchizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster,and the C-terminal half ofG. gallus,respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POLγ like activity from mitochondrial extracts. The human DNA POLγ is unique in that the first exon of the gene contains a CAG₁₀trinucleotide repeat.     

DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

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.     

Comparative analysis of in vivo interactions between Rev1 protein and other Y-family DNA polymerases in animals and yeasts

Eukaryotes are endowed with multiple specialized DNA polymerases, some (if not all) of which are believed to play important roles in the tolerance of base damage during DNA replication. Among these DNA polymerases, Rev1 protein (a deoxycytidyl transferase) from vertebrates interacts with several other specialized polymerases via a highly conserved C-terminal region. The present studies assessed whether these interactions are retained in more experimentally tractable model systems, including yeasts, flies, and the nematode C. elegans. We observed a physical interaction between Rev1 protein and other Y-family polymerases in the fruit fly Drosophila melanogaster. However, despite the fact that the C-terminal region of Drosophila and yeast Rev1 are conserved from vertebrates to a similar extent, such interactions were not observed in Saccharomyces cerevisiae or Schizosaccharomyces pombe. With respect to regions in specialized DNA polymerases that are required for interaction with Rev1, we find predicted disorder to be an underlying structural commonality. The results of this study suggest that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another.     

Damage repair DNA polymerases Y

The newly found Y-family DNA polymerases are characterized by low fidelity replication using an undamaged template and the ability to carry out translesion DNA synthesis. The crystal structures of three Y-family polymerases, alone or complexed with DNA and nucleotide substrate, reveal a conventional right-hand-like catalytic core consisting of finger, thumb and palm domains. The finger and thumb domains are unusually small resulting in an open and spacious active site, which can accommodate mismatched base pairs as well as various DNA lesions. Although devoid of a 3'-->5' exonuclease activity, the Y-family polymerases possess a unique "little finger" domain that facilitates DNA association, catalytic efficiency and interactions with auxiliary factors. Expression of Y-family polymerases is often induced by DNA damage, and their recruitment to the replication fork is mediated by beta-clamp, clamp loader, single-strand-DNA-binding protein and RecA in Escherichia coli, and by ubiquitin-modified proliferating cell nuclear antigen in yeast.     

Mitochondrial DNA polymerases from yeast to man: a new family of polymerases

We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases γ (Pol γ). Multiple amino acid sequence alignments with Pol γ from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.     

The DNA polymerase domain of pol(epsilon) is required for rapid,efficient, and highly accurate chromosomal DNA replication,telomere length maintenance,and normal cell senescence in Saccharomyces cerevisiae

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases.

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

Molecular cloning, sequence and expression of Aa-polB , a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol γ from yeast to human. Received: 13 October 1998 / Accepted: 21 December 1998     

Cloning, expression and characterization of human tissue-specific DNA polymerase λ2*

DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-³²P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.