DNA Polymerases from Bakers’ Yeast*

Two DNA polymerases are present in extracts of commercial bakers’ yeast and wild type Saccharomyces cereuisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAE-cellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (>lOO,OOO) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties DNA polymerases for distinguishing Enzyme II catalyzes pyrophosphate reactions, and has an associated 3’-exonuclease activity.

Two DNA polymerases are present in extracts of commercial bakers' yeast and wild type Saccharomyces cereuisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAEcellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (>lOO,OOO) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties of yeast DNA polymerases provides additional evidence for distinguishing the two species. Enzyme I has no associated nuclease activity but does carry out pyrophosphate exchange and pyrophosphorolysis reactions. Enzyme II catalyzes pyrophosphate exchange and pyrophosphorolysis reactions, and has an associated 3'-exonuclease activity. Enzyme I does not degrade deoxynucleoside triphosphates and cannot utilize a mismatched template. Enzyme II does carry out a template-dependent deoxynucleoside triphosphate degradation reaction and can excise mismatched 3'-nucleotides from suitable template systems. Earlier studies have shown that both Enzyme I and Enzyme II are inhibited by N-ethylmaleimide. The yeast enzymes are not identical to any known eukaryotic or prokaryotic DNA polymerases. In general, Enzyme I appears to be most similar to eukaryotic DNA polymerase LY and Enzyme II exhibits properties of prokaryotic DNA polymerases II and III.
Multiple DNA polymerases are present in both prokaryotic (1, 2) and multicellular animal cells (3)(4)(5). It is a matter of theoretical and technical interest to analyze detailed characteristics of DNA polymerases in a variety of biological systems. Analysis of DNA polymerases in simple eukaryotes (5 10) and plants (11) showed the absence of DNA polymerase /3 activity in these systems. In the limited data available (where partial purification and characterization of the DNA polymerases had been carried out) the properties of the enzymes seemed to vary with the species examined (6-9, 11). The purpose of this study was to compare certain properties of highly purified yeast DNA polymerases with other well characterized DNA polymerases from bacterial and mammalian sources (l-4, 12, 13 Yeast is of special interest because preliminary studies indicated that fungi did not show a typical eukaryotic pattern of DNA polymerase activity (5). Although the absence of DNA polymerase p in yeast (6, 7) suggests that yeast may not be a model system for studies on DNA replication and repair in more complex eukaryotic cells, the presence of a well characterized genetic system should allow analysis of biological effects through mutation. The first question concerns whether either of the yeast DNA polymerases resembles the mammalian (3, 4) or bacterial enzymes (1, 2). A notable major difference between the bacterial and mammalian enzymes is that all bacterial DNA polymerases have associated exonuclease activity (1, 2) while the mammalian DNA polymerases do not exhibit this property (12,13). Earlier studies by Wintersberger (6) and Helfman (14) showed that an exonuclease activity copurified with yeast DNA polymerase II. The results from this study confirm that yeast DNA polymerase I has no associated nuclease activity while yeast DNA polymerase II has an associated exonuclease activity. The exonuclease associated with yeast DNA polymerase II is a 3'-exonuclease and is capable of removing replication errors (1). Other properties of the yeast enzymes are also described. MATERIALS AND Step 2: Protamine Sulfate Precipitation-One liter of 2.5% protamine sulfate solution was added to 4 liters of Fraction I with stirring. The extract was allowed to stand for 30 min and was then clarified by centrifugation for 30 min at 8000 rpm in the GS-3 rotor in the Sorvall centrifuge.
The volume of the supernatant, Fraction II, was 3.9 liters.
Step 3 Step 4: Phosphocel l ul ose Column Chromatography-Fraction III was diluted with 2700 ml of 50 mM potassium phosphate at pH 7.2 and 5 mM 2-mercaptoethanol and loaded onto a phosphocellulose column (4.5 x 45 cm) previously equilibrated with the same buffer. The column was washed with 3 liters of the same buffer, and proteins were eluted with 7.6 liters of a 0 to 0.5 M linear gradient of KC1 in 50 mM potassium phosphate at pH 7.2 and 5 rnM 2-mercaptoethanol. Active fractions (1820 ml) were pooled and labeled Fraction IV. To concentrate this fraction, it was precipitated at 60% (NH&SO, saturation, collected by centrifugation, redissolved in 20 mM potassium phosphate at pH 7.2,5 rnM 2-mercaptoethanol, and 10% glycerol, and dialyzed against 2 changes of 2 liters of the same buffer. The final volume of concentrated Fraction IV was less than 100 ml. Step 5: DE11 Cel l ul ose Column Chromatography -Dialyzed, concentrated Fraction IV was loaded onto a DE11 cellulose column (2.6 x 60 cm) previously equilibrated with 25 mM potassium phosphate at pH 7.2, 5 mM 2-mercaptoethanol, and 10% glycerol. The column was washed with the same buffer and eluted with a 2.8-liter linear gradient of 0 to 0.5 M KC1 in 25 rnM potassium phosphate at pH 7.2, 5 mM 2-mercaptoethanol, and 10% glycerol. Two peaks of activity were obtained from this column (cf. Fig. 1 VIIb was fractionated on a denatured DNAcellulose column (0.9 x 2 cm) as described for Fraction VIIa except that a 60-ml gradient was used. DNA polymerase activity eluted from the column in a single peak at 0.11 M KCl. Active fractions were pooled and labeled Fraction VIIIb.

Fractions
VIIIa and VIIIb were dialyzed against 50 mM potassium phosphate at pH 7.2, 5 rnsr 2.mercaptoethanol, and 50% glycerol. The enzyme preparations were stored at -20" and the activities were stable for over 2 months.

DNA Polymerases in Wild Type Yeast Cells
Although commercial yeast cake provides a convenient source for the purification of DNA polymerases, it is essential to establish that the enzymes present are the same as those found in wild type Saccharomyces cerevisiae grown in defined media and under sterile conditions. Independent confirmation of the presence of a minor activity (DNA polymerase II) is particularly important. The conditions used in comparing the crude extract from the wild type yeast cells and commercial yeast cells were similar with two minor exceptions. Phenylmethylsulfonyl fluoride, a protease inhibitor, was included in the extraction buffer used with wild type cells. A lower final concentration of protamine sulfate was used for wild type extracts since the extracts were more dilute.
The specific activity of DNA polymerase activity (7.7 units/ mg of protein) in the crude extract of the wild type yeast cells was comparable to that of the crude extract of commercial yeast (5.5 to 10 unitslmg in various preparation). Partial purification of the wild type extract appears to proceed much as for the extract of commercial yeast (see below). For example, 7,800 units out of 17,000 units were recovered after the phosphocellulose column. A net purification of about 25-fold was obtained prior to chromatography on DE11 column. All activity found in the phosphocellulose fraction of the wild type extract was loaded onto the DE11 column and the DNA polymerase activities eluted from DE11 by a salt gradient are shown in Fig. 1. Two peaks of DNA polymerase activity are eluted from DE11 column of the wild type extract, comparable to results obtained with extracts of commercial yeast. Enzyme I, eluting at about 0.12 M KCl, was totally inhibited by the antiserum elicited by Enzyme I purified from the commercial yeast. Enzyme II (less than 10% of the total activity), eluting from the column at about 0.2 M KCl, was not inhibited by the antiserum against Enzyme I. The results of the more extensive purification procedure applied to extracts of commercial yeast are summarized in Table I. DNA polymerase I was purified 20,000-to 65,000-fold by this procedure, while DNA polymerase II was purified 8,000-to 13,000-fold. Further research will be required for preparation of homogeneous enzyme in good yield. The removal of nucleic acids from crude extract by protamine sulfate precipitation is essential for the success of the purification. The A,,, to A,,+,) ratios in the crude extract, protamine sulfate supernatant, and the dialyzed (NH&SO, fraction were 0.6, 1.    provide additional evidence for the presence of at least two distinct species of enzyme in yeast. The results obtained in this study are comparable to those obtained by others and provide some extension of earlier work. The absolute level of total DNA polymerase activity present in the crude extract described here was about 300 units/g of cells wet weight. This level is comparable to the 300 to 600 units/g of yeast previously reported by Wintersberger (6,28). The relative levels of Enzymes I and II appear to be quite different. In the preparations described in this communication, Enzyme II accounted for less than 10% of the total enzyme activity in the crude extract. In the preparation described by Wintersberger (6) Enzyme II accounted for about 30% of the total enzyme activity in the crude extract. The difference in the levels of Enzyme II could be accounted for by differences in the reaction conditions used or by the methods used for extraction of the enzyme.
The total amount of DNA polymerase activity found in commercial yeast cells appears to be somewhat greater than that in wild type yeast cells grown in the laboratory. This apparent difference is due mostly to the difference in water content of cell pellets and commercial yeast. When the levels present in lyophilized preparations were compared, the laboratory grown wild type cells contained 2,600 units/g while commercial yeast had 4,700 units/g. Although the higher level of enzyme activity in commercial cells is somewhat surprising, this finding is consistent with the observation of DNA polymerase levels in Dictyostelium discoideum. In D. discoideum, DNA polymerase level was found to be higher in stationary cells than log phase cells (10). A trivial alternative explanation could be that more efficient extraction of the commercial yeast was obtained.
The enzyme preparations described in this communication are not homogeneous. The specific activities of the preparations obtained in this laboratory were 4-to lo-fold higher for Enzyme I and 2-to 3-fold higher for Enzyme II when compared to Wintersberger's preparations. Although Enzyme II is not homogeneous, the data presented strongly suggest that the DNA polymerase and 3'-exonuclease functions are on the same enzyme protein. A definitive statement concerning the relationship of these two enzyme activities awaits the availability of homogeneous yeast DNA polymerase II.
The biological roles of the two yeast DNA polymerases are unknown at the present. It is interesting to note that in yeast where DNA polymerase p is absent (5-71, yeast DNA polymerase II with error-correcting capability can be found. The role of DNA polymerase /3 in mammalian cells has been postulated to be in DNA repair and the role of DNA polymerase cx has been postulated to be in DNA replication (3). Whether yeast DNA polymerase I indeed has a biological role similar to DNA polymerase (Y and yeast DNA polymerase II has a role similar to DNA polymerase CY remain to be demonstrated. The genetic make-up of yeast is well defined and a number of DNA synthesis and DNA repair mutants are available (29, 30). A detailed biochemical and immunological characterization of yeast DNA polymerases should facilitate the studies on the biological roles of these enzymes and should also aid in our understanding of the molecular defects of these mutants.