DNA polymerase 6 from embryos of Drosophila melanogaster

We have purified a DNA polymerase activity from 0to 2-hr embryos of Drosophila melanogaster to near homogeneity. The purified enzyme consists of a single 120-kDa polypeptide, which contains polymerase and 3'-.5' exonuclease activities. Exonuclease activity is inhibited by deoxynucleoside triphosphates, suggesting that the polymerase and exonuclease activities are coupled. The polymerase is more active with poly(dA-dT) than with activated DNA or poly(dA)/olgo(dT) as template. It shows a low degree of processivity with poly(dA)/oligo(dT). The polymerase is sensitive to aphidicolin and carbonyldiphosphonate but resistant to N2-[p-(n-butyl)phenylJ-2-deoxyguanosine triphosphate, 2-[p-(n-butyl)anilinoJ-2-deoxyadenosine triphosphate, and dideoxythymidine triphosphate. The 120-kDa polypeptide can be distinguished from the large subunit of Drosophila DNA polymerase a on the basis of the peptides generated by partial cleavage with N-chlorosuccinlmide and by its failure to react with a monoclonal antibody directed against the large subunit ofDNA polymerase a. TheDNA polymerase is inhibited by 200 mM NaCl and is unable to use poly(rA)/oligo(dT) as a template, thus differentiating it from DNA polymerase y. On the basis of these properties, we propose that the DNA polymerase that we have purified from 0to 2-hr Drosophila melanogaster embryos is DNA polymerase & At least five different DNA polymerases, a, 13, 'y, 8, and E, have been identified in eukaryotic cells (1). Polymerase a (Pol a), which contains a tightly associatedDNA primase activity, is required for nuclear DNA replication (2-6). Pol ,B is believed to function mainly in DNA repair (7). Pol y is the mitochondrial DNA polymerase and is presumably responsible for mitochondrial DNA replication (8). Pol 8 is also involved in nuclear DNA replication (3, 9, 10) and possibly in DNA repair (11). Pol e may be involved in DNA replication and repair (12-15). Pol a is composed of four subunits: DNA polymerase activity is associated with the 180-kDa subunit and primase activity is associated with the 60and 50-kDa subunits (1). A cryptic 3'--5' exonuclease activity associated with the polymerase subunit ofDrosophila Pol a appears upon separation of the 182-kDa subunit from the fourth, 73-kDa subunit (16). A 3'-*5' exonuclease activity is also associated with the immunoaffinity-purified catalytic subunit of yeast Pol a (17) and with the human lymphocyte Pol a-primase (18) but is not found in other Pol a preparations (6). Pol a is sensitive to inhibition by aphidicolin, and the nucleotide analogs N2-[p(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(nbutyl)anilino]-2-deoxyadenosine triphosphate (BuPdGTP and BuAdATP), but is resistant to inhibition by dideoxythymidine triphosphate (ddTTP) (3). Pol 6, which consists of a single -40-kDa polypeptide, is the smallest of the five DNA polymerases (1). Pol ycontains two subunits (125 and 35 kDa) and has an associated 3'-*5' exonuclease (1, 5, 19). It is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. resistant to inhibition by aphidicolin and high salt but is sensitive to ddTTP (3). It can also utilize various templates including poly(rA)/oligo(dT) (20). Pol 8 and Pol e both possess 3'-*5' exonuclease activity. However, they can be distinguished by their structure and activity (9, 10, 21). Pol 8 consists of a catalytic polypeptide (-125 kDa) and another polypeptide (=50 kDa) of unknown function. Pol E contains a catalytic subunit of >215-250 kDa and several other subunits ranging in size from 30 to 80 kDa (5). Pol 8 requires an accessory protein, proliferating cell nuclear antigen (PCNA) (22, 23), for processive DNA synthesis on templates with long single-stranded DNA (ssDNA) segments-e.g., poly(dA)/oligo(dT) with a low primer to template ratio (24, 25). Two forms ofPol 8 have been isolated from mouse cells. One consists ofa 125and a 48-kDa subunit and is stimulated by PCNA. The second contains only the 125-kDa polypeptide and does not respond to PCNA, suggesting that the 48-kDa subunit is required for interaction of Pol 8 with PCNA (26). Although the processivity of Pol E is generally independent of PCNA (27, 28), conditions can be found where Pol e is stimulated by PCNA (29, 30). Pol 8 and Pol e show different template preferences: poly(dA-dT) is the preferred template for Pol 8 and poly(dA)/oligo(dT) for Pol e (21). Pol 8 and Pol e also respond differently to various inhibitors. Pol 8 is more sensitive to inhibition by carbonyldiphosphonate and is less sensitive to inhibition by 10%o dimethyl sulfoxide (DMSO) and 0.5 mM ddTTP than Pol e (27). Both polymerases are as sensitive to aphidicolin as Pol a, but, in contrast to Pol a, are resistant to BuPdGTP and BuAdATP (27). The rapid rate of DNA replication during early embryogenesis (31) makes Drosophila melanogaster a rich source of replication enzymes, and, indeed, Pol a and Pol y have been purified from Drosophila embryos in substantial quantities (20, 32). In contrast, it has been difficult to detect either Pol 8 or Pol e in Drosophila embryos. However, Peck et aL (33) recently observed a low level of a DNA polymerase activity in Drosophila embryos that was clearly different from Pol a and is very likely Pol 8. In this paper, we describe the purification of Pol 8 from 0to 2-hr Drosophila embryos to >95% homogeneity. Unlike Pol 8 purified from mammalian cells or yeast, it consists of only a single 120-kDa polypeptide and is unresponsive to PCNA. MATERIALS AND METHODS Materials. Unlabeled dNTPs and ddTTP were from Pharmacia, and [a-32P]dTTP was from Amersham. BuPdGTP, BuAdATP, and carbonyldiphosphonate were gifts from G. E. Wright (University of Massachusetts Medical School). Abbreviations: Pol a, /3, y, 8, and e, DNA polymerase a, I, y, 8, and E; BuPdGTP, N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate; BuAdATP, 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate; ddTTP, dideoxythymidine triphosphate; PCNA, proliferating cell nuclear antigen; DMSO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; ssDNA, single-stranded DNA; DTT, dithiothreitol.