Three Forms of DNA Polymerase from Drosophila melanogaster Embryos

Abstract

DNA polymerase was purified from Drosophila melunoguster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, 11, and 111, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)z and poly(rA) . (dT)z. At the final step of purification, enzymes I, 11, and 111 were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)x.The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and I1eluted as distinct peaks and enzyme 111 eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5 -7.3 S, enzyme I1 sedimented at 7.3 -8.3 S, and enzyme 111 sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme 111, which was inactive with poly(rA) . (dT)z. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)x activity from poly(rA) . (dT)z activity.The DNA polymerase preferred poly(dA) . (dT)x (with MgZ+) as a primer-template, although it was also active with poly(rA) . (dT)x (withMn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)x was inhibited by K + and activity with poly(rA) . (dT)E was stimulated by K + and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophilu embryos have physical properties similar to those of DNA polymerase-a and enzymatic properties similar to those of all three vertebrate DNA polymerases.Prokaryotic cells [l, 21 and eukaryotic cells [3 -61 contain several DNA polymerases. The roles of DNA polymerases I, 11, and I11 of Esclzerichiu coli are being investigated by a combination of genetic and biochemical techniques [2,7]. However, the roles in vivo of the vertebrate DNA polymerases a, p, and y have not been determined. Several lines of evidence suggest that DNA polymerase-a may be the primary replication enzyme in vivo [3,5]. Proof of the functions of the eukaryotic DNA polymerases would be facilitated by

Replication of Eukaryotic Chromosomes: A Close-up of the Replication Fork

“…In Drosophila, proteolysis of a-polymerase (mol wt 280,000 ) generates three smaller enzymatically active forms (39,98,99). In mammalian cells, alterations in chromatographic procedures (67) or treatment of cells with cycloheximide (100) generates different forms of a-polymerase.…”

Association of Mutations in Replicative DNA Polymerase Genes with Human Disease: Possible Application of Drosophila Models for Studies

“…The requirement to replicate the entire genome during the very short early cycles in Drosophila embryos necessitates the rapid availability of large amounts of replication factors, including DNA polymerases. This led to the DNA polymerase α from Drosophila being one of the earliest eukaryotic replicative polymerases to be purified and characterised biochemically [35][36][37][38][39][40][41]. These early preparations of the enzyme suffered considerable proteolysis, but the adjustment of purification conditions allowed purification of an intact enzyme [42][43][44][45][46] which was observed to contain four subunits with sizes of 182 kDa (POLA1), 73 kDa (POLA2), 60 kDa (PRIM1) and 50 kDa (PRIM2) (Table 1).…”

Purification and characterization of two forms of DNA polymerase .alpha. from HeLa cell nuclei

“…As for one of the essential factors in DNA replication, DNA polymerase, DNA polymerase a is suggested to be involved in DNA replication among the three DNA polymerases, a, ß, and 7, found in mammalian cells (Weissbach, 1977). In various eukaryotic cells, the existence of multiple forms of DNA polymerase a, which differ in their chromatographic behavior, has been demonstrated (Yoshida et al, 1974;Hachmann & Lezius, 1975; Craig & Keir, 1975;Matsukage et al, 1976;PedraliNoy & Weissbach, 1977;Nishioka et al, 1977;Bieri-Bonniot & Schuerch, 1978;Hesselewood et al, 1978;Brakel & Blumenthal, 1978;Chen et al, 1979;Lamothe et al, 1981). We have also reported that HeLa cells or their isolated nuclei contain two active forms of DNA polymerase a (Ono et al, 1978(Ono et al, , 1979Tanuma et al, 1980).…”