Purification and Properties of DNA Polymerases from Chick Embryo

Abstract

The main DNA polymerases present in cytoplasmic and nuclear soluble extracts of sevenday-old chick embryos have been purified. One of them, DNA polymerase I, mainly present in the cytoplasm has been obtained about 50-60°/, pure. Its molecular weight is 148000 by Sephadex filtration and its s value is about 7.5 S. This DNA polymerase is devoid of DNAase and RNAase activity. DNA polymerase 11, of low molecular weight (50000), sedimenting a t 3 S, is the main DNA polymerase present in the nucleus and has been obtained goo/, pure. The DNA polymerase I1 activity is also present in the cytoplasm. The purified cytoplasmic DNA polymerase I1 has a molecular weight of 27000 and appears as a monomer of nuclear DNA polymerase 11. The DNA polymerase I1 preparations, from either nucleus or cytoplasm, are devoid of nuclease activity and exhibit the same primer specificity; both are able to copy RNA strands in rAn * dTn hybrids whereas DNA polymerase I only copies the DNA strands in such hybrids.Three DNA polymerases have been purified from bacteria and two, probably the major enzymes, have been isolated from animal cells [l-51. The use of mutants in the biochemical study of DNA replication or repair has greatly contributed to the progressive understanding of the physiological role of these bacterial enzymes [6,7].I n the absence of similar methods, experimental approaches of this problem in animal cells are more difficult. Using purified enzymes, the study of their activity in the presence of various "primers" might furnish valuable information on their function in the cell. I n a preliminary note we have described the partial purification [8] and more recently the template requirements [9] of a lowAbbreviations. Following Chang and Bollum [l] in this paper the terms template, initiator and primer are used. Template = polynucleotide chain used for selection of the complementary deoxynucleotide triphosphate. Initiator = oligo or polynucleotide chain complexed with the template and containing a free 3' OH end. Primer = templateinitiator complex. rA, = polyadenylic acid; dT, = polythymidilic acid; dA, = polydeoxyadenylic acid; d(A-T), = alternating deoxyadenylate and deoxythymidilate chains; dI, = polydeoxyinosinic acid; dG, = polydeoxyguanydilic acid; dC, = polydeoxycytydilic acid; A C [~H ] P~~-~R N A = a~etyl-[~H]phenylalanyl-tRNA; Sephadex CM-50 = carboxymethyl-Sephadex (3-50.

Comparative Studies on the Template‐Initiator Requirements of the Chick‐Embryo DNA Polymerase I and the Avian‐Myeloblastosis‐Virus DNA Polymerase

“…The results reported here and in other papers [13,22,231 show that in appropriate conditions with only one primer the two main chick embryo DNA polymerases and the AMV DNA polymerase can be clearly distinguished. In the presence of rA, -dTn the low-molecular-weight DNA polymerase activity polymerized dTMP especially in the presence of Mn2+ and is unable to use the RNA of the hybrid as initiator.…”

“…Seven-day-old white Leghorn chick embryos were used for the purification of cellular DNA polymerase I [13]. Avian myeloblastosis virus was a generous gift of Dr J.…”

“…Ammonium or sodium ions had a similar effect. When assays were carried out in the presence of dAn * dTn the optimal KC1 concentration was 40mM [13]. I n contrast, DNA polymerase I1 required 0.15M KC1…”

“…We have previously shown [13,22,23] that chick embryo DNA polymerase I1 catalyzed dTMP polymerization using rAn * dTn as primer in the presence of Mn2+. The results reported in Table 4 indicate that in similar conditions DNA polymerase I was unable to transcribe RNA strand; it did however catalyze dAMP polymerization using dT, and not rAn as template.…”

“…A DNA untwisting enzyme has been purified from mouse cells [lo] and resembles the LO protein from E. coli [ll]. Ribonuclease H enzymes purified from bacteria and from eukaryotes show similar catalytic properties I n order to obtain information as to whether the basic mechanisms involved in DNA replication in bacteria and in higher cells are similar, the properties of chick embryo DNA polymerase I purified from the cytoplasmic compartment have been investigated [13]. The results reported in this paper show that this enzyme acts as a repair DNA polymerase and resembles DNA polymerase I11 of E. coli by [6-81. its sensitivity to high salt concentrations [14].…”