Umberto Bertazzoni

Eukaryotic DNA polymerases

Abstract The biological properties, classification and phylogeny of eukaryotic DNA polymerases are reviewed.

The poly A content and secondary structure of the 14S calf lens messenger RNA

Summary The 14S calf lens messenger RNA (mRNA) coding for the A2 chain of α-crystallin was isolated from calf lens polyribosomes by zonal centrifugation and purified by poly-(dT)-cellulose chromatography and subsequent sucrose gradient centrifugation. From the purified messenger the poly A content was estimated by measuring the increase of the fluorescence of Ethidium-bromide intercalating the hybrid formed by titration with poly U. * About 15% of the messenger is composed of poly A. The characteristics of the poly U titration curves indicate that other bases are also present in the poly A track. Further the 14S messenger exhibits a secondary structure comparable with TMV and Globin mRNA.

Variation in DNA ligase structure during repair and replication processes in monkey kidney cells.

Using a method that detects catalytically active DNA ligase in NaDodSO4-polyacrylamide gels (activity gels) we have characterized ligase produced in CV1-P monkey kidney cells infected with SV40 or treated with mitomycin C. Purification on hydroxylapatite columns of DNA ligase from control cells results in two peaks of activity called ligases I and II, respectively. Analysis of ligase I on activity gels revealed major catalytic peptides with Mr of 120, 110, 70 and 58 kDa, while analysis of ligase II revealed two major peptides of 65 and 58 kDa. Infecting CV1-P cells with SV40 produced a significant increase in the 120, 110, 70 and 58 kDa peptides while treating them with mitomycin C produced a significant increase in the 70 and 58 kDa peptides and a decrease in the 120 and 110 kDa ones. Autoproteolysis of partially purified ligase under several conditions resulted in an increase in the 58 kDa peptide and in the disappearance of other peptides. These results suggest that at least one active polypeptide is common to ligases I and II.

Radioactive labeling and analysis of 3′-terminal nucleotides of DNA fragments

Abstract 3′-Hydroxy oligodeoxyribonucleotides (average chain length 54 nucleotides), prepared by digestion of calf thymus DNA with spleen acid deoxyribonuclease followed by dephosphorylation, were used as primers for the addition of two ribonucleotides, using terminal deoxyribonucleotidyl transferase and [α-32P]ATP as donor. Conditions for the optimal incorporation of labeled nucleotides were investigated. A method for the separation of the four deoxyribonucleotides and of AMP on DEAE-cellulose columns was devised. This permitted the analysis of 3′-terminal deoxyribonucleotides after spleen exonuclease digestion of the 32P-labeled oligodeoxyribonucleotides, thus avoiding the alkaline splitting of the inter-ribonucleotide bond and the phosphatase digestion which would otherwise be necessary. The results obtained by this procedure were found to be in excellent agreement with the analysis of the 3′-terminal nucleosides released from the same DNA fragments, thus showing that the use of terminal transferase provides a valid method for determining the composition of 3′-terminal nucleotides.

DNA Ligase Activity in Human Cells from Normal Donors and from Patients with Bloom’s Syndrome and Fanconi’s Anemia

DNA ligase was analyzed in human cells obtained from normal individuals and from Bloom’s syndrome (BS) and Fanconi’s anemia (FA) patients. The level of enzyme activity in untrans-formed and transformed FA cell lines was identical to that of control cells while it was significantly higher in BS cell lines. These results were confirmed after purification of the BS or control cell enzymes on fast protein liquid chromatography from crude extracts and from ammonium sulfate pellets. After precipitation with polymin-P a lower level of ligase activity was found in BS cells. The structural property of the enzyme, investigated by activity gel analysis, showed that active polypeptides of identical sizes are obtained from control or BS cell lysates or extracts and that ligase activity could be recovered from the polymin-P pellet of BS cell extracts. These results indicate that FA and BS do not appear to be deficient in DNA ligase activity.

Activity Gels of Poly(ADP-Ribose) Polymerase: Phylogenetic Studies and Variations in Human Blood Cells

We have devised an activity gel procedure to recover the functional poly(ADP-ribose) polymerase catalytic activities after SDS-polyacrylamide gel electrophoresis [1]. Using this technique, we analyzed the catalytic polypeptides of this enzyme present in extracts obtained from a variety of organisms, belonging to a wide evolutionary scale. We also measured the enzyme in human lymphocytes before and after stimulation with mitogens and in human cells of different leukemia states.

Identity between Nuclear and Mitochondrial DNA Polymerases γ from Chick Embryo

The availability of specific tests for DNA polymerases α and β has made possible to obtain quantitative determinations of these enzymes in crude extracts and to study their physiological roles (1). A new specific assay for DNA polymerase γ has been described recently by the Weissbach group (2) who showed that while γ-polymerase is active on poly(A)·oligo(dT) in the presence of phosphate, β-polymerase is completely inhibited. This has opened up the possibility to distinguish this enzyme from α and β-polymerases and thus to study its localization and function.

Functions of DNA Polymerases α, β and γ in DNA Replication and Repair

The cells of vertebrate organisms contain three distinct DNA polymerases which have been designed α, β and γ -polymerases (1). The information concerning the physical, chemical and catalytical properties of these enzymes has progressed considerably during the past few years so that the distinction of their activities is easily obtained by utilizing the differences in molecular weight, sensitivity to inhibitors, chromatographic elution, ability to copy various templates (reviews: 2–8). The general properties of the three enzymes are summarized in Table I.