Active Pol Research Articles

DNA-Polymerases α, δ and ε from T-Cell Spontaneous Lymphoblastic Leukemia of Sprague-Dawley Inbred Rat: Isolation and Characterization

Using a single isolation procedure and selective assays for the determination of enzyme activity, highly purified DNA-polymerases α, δ and ε were isolated from the lymphoma of Sprague-Dawley inbred rats. For pol α the subunit composition was 170, 70, 57 and 53 kDa with sedimentation coefficient 8.7 S for the native molecule; pol delta consists of two polypeptides (133 and 46 kDa; sedimentation coefficient 8.2 S), while pol ε is a single polypeptide (140 kDa) and its sedimentation coefficient is 7.0 S. Comparison of the interaction of individual enzymes with known inhibitors and proliferating cell nuclear antigen (PCNA) using the template-primer poly dA-oligo dT12-18, gave the following data: (i) pol α is selectively inhibited by N2-(p-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and stimulated by dimethyl sulfoxide; (ii) all the enzymes are inhibited by N-ethylmaleimide and aphidicolin; (iii) PCNA stimulates pol δ approximately 50 times while pol ε is moderately inhibited; (iv) pol α exhibits considerably higher DNA-primase activity with poly dC as template than with poly dT, and negligible 3'-5'-exonuclease activity whereas pol δ and pol ε, which do not exert any DNA-primase activity have approximately the same 3'-5'-exonuclease activity. The ability of individual polymerases to utilize poly dT-oligo dA12-18 as a template-primer at different pH values, ionic strengths and Mg2+-concentrations was also investigated. In comparison to poly dA-oligo dT12-18 template-primer, pol α has 140% of enzyme activity on poly dT-oligo dA12-18 under optimal conditions, whereas the activity of pol ε and pol δ is 4 times and 10 times lower, respectively.

Effect of Single DNA Lesions on in Vitro Replication with DNA Polymerase III Holoenzyme : Comparison with other Polymerases

In the present work, we have studied in vitro replication of N -2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II ( cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the α subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G 1G 2CG 3CC-) have been used in primer extension assays. Site-specifically platinated 5′d(ApG) or 5′d(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the Nar I site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3′→5′ proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf(exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.

Interchangeable RNA polymerase I and II enhancers.

The RNA polymerase I (pol I) enhancer of Saccharomyces cerevisiae contains at least three elements commonly associated with RNA polymerase II (pol II) enhancers, binding sites for the transcriptional activators general regulatory factor 2 and autonomously replicating sequence-binding factor I, and a thymidine-rich element. When the particular form of the thymidine-rich element found in the pol I enhancer was placed in front of a pol II promoter, transcription was stimulated 43-fold, comparable to the effect of a powerful pol II activator such as Gal4. Conversely, when two copies of a thymidine-rich element from a pol II enhancer were placed upstream of a pol I promoter, transcription was stimulated 38-fold. This functional reciprocity of pol I and II enhancers may reflect similarities in the mechanisms of transcriptional activation. The pol I enhancer also contains an element that appears to be pol I-specific and prevent the activation of pol II.

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-define dificiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either polA1 or recA4412 mutation causing difeiciency of the polymerase activity of pol I are unable to grow at 42°C on minimal medium supplemented with adenine, i.e., when the SOS response is continuosly induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA sythesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5′-3′ exonuclease activity of pol I showss normal growth under coonditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.

Sensitivity of polA mutants of Escherichia coli K-12 to ozone and radiations.

Different polA mutants of Escherichia coli K-12 were exposed to ozone, X-rays and UV radiation, in order to compare the role of the various enzymatic activities of DNA polymerase I in the repair of damage caused by these agents. As was the case with radiations, the polymerase-deficient mutants were the most sensitive to ozone, followed by the 5'-3' exonuclease-deficient mutants. The 3'-5' exonuclease activity of pol I appeared to play a minor role in the repair of damage induced by all these agents.

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities.

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.

DNA polymerases of Tetrahymena pyriformis. II. Does an N-ethylmaleimide-resistant polymerase exist in Tetrahymena?

We investigated the relation between two N -ethylmaleimide(NEM)-sensitive DNA polymerases (designated as pol A and pol B in the preceding paper) and an NEM-resistant DNA polymerase (Furukawa et al . (1979) Nucl. Acids. Res . 7 , 2387–2398) of Tetrahymena pyriformis with respect to changes in activity during cell growth and to chromatographic properties. Pol A activity per culture increased in accordance with the increase in cell number during the log phase but decreased during the stationary phase. Pol B and NEM-resistant polymerase activities per culture also increased during the log phase and the increases continued even in the stationary phase. All the changes were protein synthesis-dependent, since the changes were inhibited by treating the cells with cycloheximide. The changes of pol B and NEMresistant polymerase activities during cell growth were always parallel and the ratio of the two enzymatic activities (NEM-resistant polymerase activity/pol B activity) remained constant with a value of about 0.03. This was also the case for cycloheximide-treated cultures. Furthermore, the enzymatic activities could not be separated by successive column chromatographies on phosphocellulose, DEAE-Sephadex A-25 and thiol-Sepharose. In these chromatographies, the elution profiles of the two activities coincided with each other and the activity ratio kept a constant value of about 0.03. From these results, it is likely that the apparent NEM-resistant activity we measured corresponds to the remaining activity of pol B under NEM inhibition; in other words, no NEM-resistant polymerase really exists in Tetrahymena .

Ischemic damage to the sarcoplasmic reticulum of skeletal muscles: The role of lipid peroxidation

The development of ischemia was shown to be accompanied by inhibition of the Ca2+ enzyme transport system (ETS) (a decrease in the Ca2+/ATP ratio and in activity of Ca2+-dependent ATPase), which correlates with accumulation of the primary and secondary molecular lipid peroxidation products (POL) in vivo and in the membranes of the sarcoplasmic reticulum (SR) of the skeletal muscles. Administration of antioxidants (2,6-di-tert-butyl-4-methylphenol, α-tocopherol) prevents activation of POL in the ischemic muscle and partially protects the Ca2+ ETS against injury. Restoration of the blood flow after prolonged ischemia leads to further inhibition of the Ca2+ ETS while the concentration of POL products remains unchanged.

Excision-repair in mutants ofEscherichia coli deficient in DNA polymerase I and/or its associated 5′→3′ exonuclease

The ultraviolet (UV) sensitivity of Escherichia coli mutants deficient in the 5' leads to 3' exonuclease activity of DNA polymerase I is intermediate between that of pol+ strains and mutants which are deficient in the polymerizing activity of pol I (polA1). Like polA1 mutants, the 5'-exonclease deficient mutants exhibit increased UV-induced DNA degradation and increased repair synthesis compared to a pol+ strain, although the increase is not as great as in polA1 or in the conditionally lethal mutant BT4113ts deficient in both polymerase I activities. When dimer excision was measured at UV doses low enough to avoid interference from extensive DNA degradation, all three classes of polymerase I deficient mutants were found to remove dimers efficiently from their DNA. We conclude that enzymes alternative to polymerase I can operate in both the excision and resynthesis steps of excision repair and that substitution for either of the polymerase I functions results in longer patches of repair. A model is proposed detailing the possible events in the alternative pathways.