Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

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.     

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages

The replication DNA polymerase (gp43) of the bacteriophage T4 is a member of the pol B family of DNA polymerases, which are found in all divisions of life in the biosphere. The enzyme is a modularly organized protein that has several activities in one polypeptide chain (900 amino acid residues). These include two catalytic functions, POL (polymerase) and EXO (3-exonuclease), and specific binding activities to DNA, the mRNA for gp43, deoxyribonucleotides (dNTPs), and other T4 replication proteins. The gene for this multifunctional enzyme (gene 43) has been preserved in evolution of the diverse group of T4-like phages in nature, but has diverged in sequence, organization, and specificity of the binding functions of the gene product. We describe here examples of T4-like phages where DNA rearrangements have created split forms of gene 43 consisting of two cistrons instead of one. These gene 43 variants specify separate gp43A (N-terminal) and gp43B (C-terminal) subunits of a split form of gp43. Compared to the monocistronic form, the interruption in contiguity of the gene 43 reading frame maps in a highly diverged sequence separating the code for essential components of two major modules of this pol B enzyme, the FINGERS and PALM domains, which contain the dNTP binding pocket and POL catalytic residues of the enzyme. We discuss the biological implications of these gp43 splits and compare them to other types of pol B splits in nature. Our studies suggest that DNA mobile elements may allow genetic information for pol B modules to be exchanged between organisms.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1489–1496.Original Russian Text Copyright © 2004 by Petrov, Karam.     

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.     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

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.     

DNA Polymerase C of the Thermophilic Bacterium Thermus aquaticus: Classification and Phylogenetic Analysis of the Family C DNA Polymerases

Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively. We have sequenced the structural gene encoding the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus. This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular weight of 137,388 daltons. Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase. The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C. Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important conclusion can be drawn. First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic. Second, the low-G+C Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II). Third, the cyanobacterial family C DNA pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria. Received: 7 October 1998 / Accepted: 12 January 1999     

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.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.     

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction. Received: January 22, 1998 / Accepted: February 16, 1998     

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.     

Molecular cloning of a gene required for DNA replication in Ustilago maydis

TSD2, a gene necessary for DNA synthesis in Ustilago maydis, was cloned by complementation of the temperature sensitive growth defect of a mutant known previously as pol1-1 and renamed here tsd2-1. Linkage analysis established that the cloned fragment contained an allele of tsd2-1 and not a suppressor. DNA sequence determination of the cloned DNA fragment indicated the presence of a single large uninterrupted open reading frame capable of encoding a protein of 845 amino acids without homology to any known gene involved in DNA synthesis. TSD2 was found to be cell cycle-regulated and mRNA levels peaked in early S or G₁ phase. Received: 27 March 1996 / Accepted: 28 August 1996     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) , and are necessary for replication of nuclear DNA. Po1 interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae

A screen for mutants of budding yeast defective in meiotic gene conversion identified a novel allele of the POL3 gene. POL3 encodes the catalytic subunit of DNA polymerase delta, an essential DNA polymerase involved in genomic DNA replication. The new allele, pol3-ct, specifies a protein missing the last four amino acids. pol3-ct shows little or no defect in DNA replication, but displays a reduction in the length of meiotic gene conversion tracts and a decrease in crossing over. We propose a model in which DNA synthesis determines the length of strand exchange intermediates and influences their resolution toward crossing over.     

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, 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.