ABSTRACT. DNA polymerase activity (DPA) was investigated in a synchronous culture of the unicellular green alga, Chlorella ellipsoidea (Tamiya's strain). During the algal cell cycle, which was observed in the synchronous growth, nuclear DNA (nDNA) is synthesized near the end of the light phase, whereas the major chloroplast DNA (chDNAM) is synthesized in the middle preceeding the synthesis of nDNA (1). The DPA on a per cell basis correlated well with the synthetic patterns of the cellular DNAs, except during the early stages. The two marked rises in total cellular DPA, thus corresponded directly to the syntheses of the two DNA species.Fractionation of DNA-depleted enzyme preparations by DEAE-cellulose column chromatography showed two kinds of DNA polymerases. One was eluted at 0.13 M-KCl (Pol I) and the other at 0.15 M-KCl (Pol II). Pol II activity (DPA-II) reached its maximum during chDNA synthesis, whereas Pol I activity (DPA-I) reached its maximum at the end of nDNA synthesis period. We concluded that Pol II synthesizes or replicates chDNA in vivo and Pol I, nDNA; hence, we have designated them "chloroplastic" and "nuclear" DNA polymerases.We also examined the inhibitory effects of chloramphenicol (CP) and cycloheximide (CH) on the development of the two DPAs during the cell cycle. DPA-II was inhibited principally by CP, and DPA-I by CH. Moreover, CH somewhat inhibited DPA-II development during the early phase, and CP greatly inhibited DPA-I development after chDNA synthesis.Thus, Chlorella cells contain at least two kinds of DNA polymerases, chloroplastic and nuclear. The former probably is synthesized on chloroplastic ribosomes and the latter on cytoplasmic ribosomes.Chlorella cells usually contain one nucleus, one chloroplast and one mitochondrion (2). A repetitive synchronous culture of the strain, C. ellipsoidea (Tamiya's strain), has been established for some time (3). We have used it, as a model system for green

Previously, we reported two DNA polymerases (DPols), Pol I and Pol II, in the unicellular green alga, Chlorella (Aoshima, J., Nishimura, T., & Iwamura, T. (1982) Cell Struc. Funct. 7, 71-86). Changes in their activities during the cell cycle either in the normal and drug-inhibited courses indicated that Pol I and Pol II functioned to replicate nuclear and chloroplast DNAs, respectively. In the present work, we have examined their enzymic properties to characterize and distinguish them further. A number of inhibitors commonly used for such studies were also tested to determine their effects and the results were analyzed by use of the simple and useful "Tamiya plot." We have also analyzed the data obtained in inhibitor studies on various eukaryotic DPols in the literature using the Tamiya plot, and the results will be presented elsewhere (Iwamura, T. & Aoshima, J. (1984) J. Biochem. in press). Comparisons of the algal DPols with mammalian enzymes as regards enzymic properties and inhibition modes have led us to conclude that: [1] the algal two DPols are significantly different from each other, despite having many similarities to each other: [2] they are related in properties to any of the three mammalian DPols-alpha, -beta, and -gamma; Pol I (n-DPol) was a little more like alpha than Pol II (ch-DPol), which in turn more akin to gamma. This feature was quantitized by using vectors in a three-dimensional alpha-beta-gamma-space. Another peculiar feature derived from the Tamiya plot of the inhibitions by araCTP and aphidicolin (both being competitive with cCTP) has led us to propose a specific allosteric role of cCTP in the reaction mechanism besides its role as one of the substrates.

In connection with the characterization of two DNA polymerases (DPols) of Chlorella, we have extensively surveyed the literature on inhibition studies on DPols in various eukaryotes. By applying Tamiya's plot (1), we have obtained two parameters for each of the inhibitors, phi- and n-values, which express the enzyme sensitivity to the drug and the number of inhibitor molecules present in the enzyme-inhibitor complex that is principally involved in the inhibition, respectively. By inspecting these parameters for the three mammalian DPols, alpha-, beta-, and gamma-pols, as well as other eukaryotic DPols, we have found that: [1] inhibitors commonly utilized for characterizing various DPols can be classified into two major groups, each having two subgroups, on the basis of a comparison of the phi values among alpha-, beta-, and gamma-pols. Moreover, the grouping seems not to be merely coincidental, but to be intrinsically related to facets of the enzyme reaction, which may be taken to reflect evolutionary differences in DPol structure and function among the three DPols; [2] the remarkable n value, n = 1/2, that has been found for the inhibitors competitive with dCTP in Chlorella DPols has also been detected widely in many other eukaryotic DPols. Based on the first finding as well as many other data on various DPols, we have proposed an evolutionary scenario for eukaryotic DPols. Based on the second finding, we have hypothesized a novel role for dCTP as a cofactor, probably an apparent allosteric effector, in the nucleotide transfer reaction mechanism.