Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.     

Determination of the DNA conformation of the simian virus 40 (SV40) enhancer in SV40 minichromosomes

The simian virus 40 (SV40) enhancer contains three 8-bp purine-pyrimidine alternating sequences which are known to adopt the left-handed Z-DNA conformation in vitro. In this paper, we have undertaken the determination of the DNA conformation adopted by these Z-motifs in the SV40 minichromosome. We have analyzed the presence of Z-DNA through the change in linkage which should accompany formation of this left-handed conformation. Our results indicate that, regardless of the precise moment of the viral lytic cycle at which minichromosomes are harvested and the condition of the transfected DNA, either relaxed or negatively supercoiled, none of the three Z motifs of the SV40 enhancer exist to a significant extent as Z-DNA in SV40 minichromosomes. The SV40 enhancer adopts predominantly a right-handed B-DNA conformation in vivo.     

UV-Irradiated SV40 minichromosomes as substrates for DNA repair endonucleases

UV-irradiated SV40 minichromosomes have been shown to be a substrate for a purified DNA repair endonuclease. A UV-repair endonuclease activity was also found to be associated with the isolated SV40 minichromosomes themselves. It appeared to have similar properties to the enzymes described from other mammalian sources.     

Replication of SV40 minichromosomes in vitro

We operationally define two forms of SV40 minichromosomes, a 75S-form, prepared at low salt concentration, referred to as native minichromosomes, and a 50S-form, obtained after treatment with 0.5M potassium acetate, the salt-treated minichromosomes. Both preparations of minichromosomes serve well as templates for replication in vitro. Their respective replication products are strikingly different: replicated native minichromosomes contain a densely packed array of the maximal number of nucleosomes whereas replicated salt-treated minichromosomes carry, on average, half of the maximal number. We conclude that in both cases parental nucleosomes are transferred to progeny DNA, and, in addition, that an assembly of new nucleosomes occurs during the replication of native minichromosomes. This is apparently due to the presence of a nucleosome assembly factor as a constituent of native minichromosomes that dissociates upon treatment with salt. We further show that preparations of minichromosomes usually contain significant amounts of copurifying hnRNP particles and SV40 virion precursor particles. However, these structures do not detectably affect the replication and the chromatin assembly reactions.     

Conditions for sliding of nucleosomes along DNA: SV 40 minichromosomes

'Sliding' of nucleosomes along DNA under nearly physiological conditions was studied using treatment of SV 40 minichromosomes with the single-cut restriction endonucleases EcoRI and BamHI. Each enzyme can convert no more than 20-25% of the circular DNA molecules of minichromosomes into the linear form irrespective of the presence of histone H1. This suggests absence of the nucleosomes lateral migration (sliding) along DNa at least in the vicinity of the restriction endonucleases cleavage sites during several hours of incubation. The sites available for EcoRI and BamHI in minichromosomes seem to be located predominantly in the spacer DNA regions of nucleosomes. Introduction of only one double-strand (but not single-strand) break into the DNA of minichromosomes stripped of histone H1 is sufficient to induce redistribution of the nucleosome core particles due to their sliding along DNA. Thus, sliding of the nucleosome core particles can be induced under physiological conditions by rather low energy expenditures.     

Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.

Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).     

Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases

With a procedure that allows the renaturation of the DNA polymerase catalytic activity in situ after SDS-polyacrylamide gel electrophoresis, we have compared the active polypeptides present in extracts from organisms covering a wide evolutionary range from prokaryotes to eukaryotes, namely: Escherichia coli, Oryza sativa, Daucus carota , Neurospora crassa, Dictyostelium discoideum, Saccharomyces cerevisiae, Ceratitis capitata, Leucophaea maderae , Xenopus laevis, rat tissues and human lymphoblastoid cells. Two main clusters of active peptides are visible in mammalian and adult insect tissues, characterized by a mol. wt. greater than 70000 and less than 50000, respectively. High mol. wt. peptides are heterogeneous in size and correspond to active fragments of DNA polymerase alpha, whereas low mol. wt. peptides show the same migration rate as purified DNA polymerase beta and are not generated by proteolysis of the high mol. wt. cluster, In the three species of fungi studied, only high mol. wt. peptides are found. The same is true in plant cells, where no DNA polymerase beta activity is detectable and the pattern of the high mol. wt. cluster is similar to that observed in E. coli extracts (which also lack low mol. wt. peptides). Also in mitochondria from higher and lower eukaryotes only high mol. wt. species are observed, and the active band(s) range from 70000 to 145000 daltons. Our results indicate that the structure of DNA polymerase has been highly conserved during evolution so that an active fragment of mol. wt. greater than or equal to 70 000 is always found in prokaryotic enzymes and in the replicative species of eukaryotic and mitochondrial DNA polymerases; at a certain stage in evolution, another species of low mol. wt. DNA polymerase (beta or beta-like) appears.     

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.