Abstract
The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase. Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively. The mitochondrial enzyme was insensitive to the drug. N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM). Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM). The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM). With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases. Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting. Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III. The results show that DNA polymerase III is distinct from DNA polymerase I and II.
Highlights
From the Department of Biological Chemistry, Diuiswn of Biology and Biomedical Sciences,Wmhington Uniuersity School of Medicine, S i Louis, M&souri63110
The inhibition data in crude extracts roughly correlate with the actual ratio of DNA polymerase III/DNA polymerase I obtained after DEAE-silica gel chromatography (Fig. 1).Theseresults show that:(i) dialyzed ammonium sulfate preparations (Fraction 11) from proteasedeficient cells contain mostly DNA polymerase 111; (ii) the amount of DNA polymerase Iwas increased when these cells were frozen and thawed prior to breakage; (iii) extracts (Fraction 11) from wild-type cells broken in the absence of proteaseinhibitorscontain mostly DNA polymerase I; (iv) because the degree of inhibition observed in crude extracts agrees well with the relative activities of DNA polymerases I11 and I observed after DEAEsilica gel high pressure liquid chromatography (HPLC) chromatography, we conclude that none of the activities is preferentially inactivated durincghromatography of this column
Since the first report in 1970 on the separation of yeast DNA polymerases I and I1 [17], numerous studies have appeared on these two enzymes [5, 6, 12, 18,19]
Summary
The rigorous prevention of proteolysis during yeast cell breakage in combination with the resolving power of high pressure liquid chromatography (HPLC)‘ has allowed us to identify and purify a novel DNA polymerase activity from log activated calf thymus DNA as a substrate; the regular exonuclease assay used 3”labeled single-stranded calf thymus DNA as a substrate [1]. Monoclonal antibodies to yeast DNA polymerase I were a gift from Dr L. Chang (Uniformed Health Services University) [8] These monoclonals were generated against the native DNA polymphase cells of Saccharomyces cerevisiae [1].DNA polymerase erase I, containing predominantly the 140-kDa polypeptide and its. None of these antibodies inhibit enzyme activity directly, but their affinity for DNA polymerase I was measured by precipitation with secondary antibodies [8]: The monoclonal antibodies do not bind to the denatured enzyme and they fail to give a signal on Western blots.
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