Isolation of the gene encoding yeast DNA polymerase I

Cell, Vol. 43, 369-377,

November

1965, Copyright

0092-6674/65/l

0 1965 by MIT

10369-09

$02.0010

Isolation of the Gene Encoding Yeast DNA Polymerase I Lianna M. Johnson,’ Michael Snyder,t Lucy M. S. Chang,* Ronald W. Davis,t and Judith L. Campbell* Divisions of Chemistry and Biology 164-30 California Institute of Technology Pasadena, California 91125 t Department of Biochemistry Stanford University School of Medicine Stanford, California 94305 *Department of Biochemistry Uniformed Service University of the Health Sciences Bethesda, Maryland 20814 l

A yeast genomic DNA expression library in kgtll and antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants. Introduction DNA replication in eukaryotic cells occurs at a defined time in the cell cycle, the S phase. During this period, replication is initiated at numerous origins of replication, each origin being used only once in what appears to be a temporally defined order (Newlon et al., 1974; Burke and Fangman, 1975). Little is known about what mechanisms control the initiation of DNA synthesis or about what proteins are involved in eukaryotic DNA replication. To understand these processes, it is essential to know what gene products are involved and how they are regulated. A genetic approach to the study of cell division, including DNA replication, was undertaken by Hartwell and coworkers in Saccharomyces cerevisiae. A series of mutations was isolated that blocked the cell division cycle at specific points (cdc mutations), and a number of these mutants were found to be defective in DNA replication (Hartwell, 1971; Culotti and Hartwell, 1971). The gene products for three of these genes have now been identified (cdc9, DNA ligase [Johnston and Nasmyth, 19781; cdcd, thymidylate kinase [Sclafani and Fangman, 1984;

Jong et al., 19841; cdc27, thymidylate synthetase [Bisson and Thorner, 19771). However, no mutant affecting the central figure in replication, DNA polymerase, was found among the mutants collected, nor among other temperature sensitive mutant collections (Johnston and Game, 1978; Dumas et al., 1982; Kuo et al., 1983). Biochemically, three DNA polymerases have been identified in S. cerevisiae. One polymerase is associated with the mitochondria, and the other two are nuclear polymerases (for review see Campbell, 1983). Both nuclear enzymes (DNA polymerase I and II) are large (>140,000 daltons) and are sensitive to the drug aphidicolin (Wintersberger and Wintersberger, 1970; Wintersberger, 1974; Chang, 1977; Badaracco et al., 1983; Plevani et al., 1985). By these two criteria they can both be categorized as cr-polymerases, the enzymes thought to be involved in eukaryotic replication. DNA polymerase I makes up 700/o-90% of the polymerase activity in the nucleus, with DNA polymerase II contributing the remaining 10%-300/o (Wintersberger and Wintersberger, 1970; Chang, 1977). Drug inhibition studies have revealed only minor differences in the two enzymes, however they have been shown to be distinct enzymes by both immunological criteria and substrate preference (Chang, 1977; Wintersberger, 1978). These latter studies indicate that DNA polymerase I uses RNA primers more efficiently than polymerase II and hence is the more likely candidate for the major replication enzyme. Furthermore, a primase activity, which synthesizes primers similar to those found in nascent DNA in higher eukaryotes (Reichard et al., 1974) can be purified in tight association with DNA polymerase I (Singh and Dumas, 1984; Plevani et al., 1984; Plevani et al., 1985). In addition, a mutant resistant to aphidicolin, a drug known to inhibit eukaryotic DNA polymerases, has been isolated in yeast (Sugino et al., 1981). DNA polymerase I purified from the mutant showed an increased resistance to aphidicolin, whereas DNA polymerase II purified from the mutant showed no change in response to aphidicolin. Thus, the weight of circumstantial evidence supports a role for DNA polymerase I in replication. However, none of the studies to date rule out the possibility that DNA polymerases I and II may have overlapping functions or may be encoded by duplicated genes. To investigate the role of DNA polymerases in yeast, we have isolated the gene encoding DNA polymerase I. Below, we describe the initial characterization of the gene, the overproduction of DNA polymerase I in both yeast and Escherichia coli, the purification of DNA polymerase I to homogeneity, and the results of disrupting the gene in vivo. Results lmmunoscreening of ngtll Yeast Genomic DNA Expression Library with Anti-DNA Polymerase I Antibody To isolate the yeast gene for DNA polymerase

I, a Agtll

a

Figure 1. Plaque Purification Polymerase I Antibody

of 1gtll

Rescreening of four potential positives One did not react with the antiserum. at a much reduced intensity.

Clones

that React

with

b

c d

DNA

with DNA polymerase antibody. Negative plaques can be seen

genomic DNA library was screened using antibodies to DNA polymerase I. Agtll is a phage expression vector that can synthesize o-galactosidase fusion proteins from the inserted DNA (Young and Davis, 1983). In a proteasedeficient E. coli strain (Lon-), these fusion proteins are generally stable enough to be detected with antibody probes. Prior to this study, DNA polymerase I had not been purified to homogeneity. However, an antiserum had been prepared against a preparation of the enzyme that is approximately 50% or greater homogeneous (Chang, 1977). This serum reacts well against immunoblots of the enzyme and strongly inhibits the activity of the enzyme in vitro (Badaracco et al., 1983). This anti-DNA polymerase I antibody was used to screen a Agtll yeast genomic DNA library (Snyder and Davis, unpublished) using modified protocols of Young and Davis (1984) and St. John and Weissman (Landau et al., 1984) as described in Experimental Procedures. One million recombinant phage were screened, and 118 positive phage were identified. Seventy-seven of these phage were purified and tested for reactivity with serum from immunized and control rabbits (Figure 1). Sixty-four clones encoding proteins that react with serum from immunized rabbits but do not react with serum from control rabbits were chosen for further study. Since the antibody used in the study reacts with several polypeptides in addition to DNA polymerase I, it was expected that clones in addition to those encoding DNA polymerase would be isolated. Several independent methods were used to identify the desired clones. The 84 clones were grouped by cross-hybridization into 6 classes. The DNA inserts from 10 clones were hybridized to plaques of all 64 clones. The 6 major classes contain: class I, 42 members; class II, 10 members; class Ill, 4 members; class IV, 3 members; class V, 3 members; class

55,000

Figure 2. lmmunoblot bodies

Analysis

Using

Class

I Affinity-Purified

Anti-

Partially purified DNA polymerase I was electrophoresed in a 7.5% polyacrylamide gel and transferred to nitrocellulose. Lane a was reacted with polymerase antisera, and lane b was reacted with affinitypurified antibodies from class I polypeptides. The antibody-antigen complexes were detected with ?-protein A. (Lanes c and d) Homogeneous DNA polymerase I was electrophoresed in a 7.5% polyacrylamide gel and was stained with either silver nitrate as described by Wray et al. (1981; lane c) or was reacted in situ in an activity gel (Spanos et al., 1981; lane d).

VI, 2 members. Class I is shown below to encode the polymerase I gene. Immunological Identification of the DNA Polymerase I Gene Immunological cross-reactivity between clone-specified polypeptides and DNA polymerase I was demonstrated by two methods. In the first approach, four mouse monoclonal antibodies that bind yeast DNA polymerase I (Plevani et al., 1984) were pooled and reacted with clonespecified polypeptides. Initially plaques were screened with the monoclonal antibodies. However, the signals observed were too weak and were not reproducible. Therefore, the reaction of the monoclonal antibodies with clonespecified antibodies was quantitated using an ELISA assay as described in Experimental Procedures. Polypeptides from class I clones showed a reproducible 2-to 3-fold higher level of reaction with the monoclonal antibodies than did polypeptides produced from the Agtll vector.

Yeast 371

DNA

Polymerase

Table

1. DNA Polymerase

I Gene

Activity

DNA Polymerase

in Plasmid-Containing

Strains

Activity”

Plasmid

First Experiment (Units)t

Second (Units)T

YEp24 pPOLt-1 DPOLl-2

5.3 23 25

8.0 34 33

Experiment

l DNA polymerase activity determined for 5 rg crude extract. Two independent transformants were assayed (first and second experiment). t 1 unit = 1 pmol total nucleotide incorporated in 1 hr at 35%.

Classes II-IV and VI showed no significant reaction with the monoclonal sera (data not shown). The second approach used for determining immunological cross-reactivity was to test whether clone-encoded polypeptides could affinity-purify antibodies that react with yeast DNA polymerase I (Snyder and Davis, 1985). Only class I was investigated because it was the largest class, because RNA blot analysis showed that this was the only class that hybridized to an RNA large enough to encode the catalytic subunit of DNA polymerase I (data not shown), and because the results of the ELISA assays looked promising. A nitrocellulose filter was saturated with a lysate of cells infected with a class I Agtll phage, and the filter was incubated with the polymerase antiserum (see Experimental Procedures). The antibodies that were not specific for class l-encoded proteins were washed off the filter, and the class l-specific antibodies were then eluted with 0.2 M glycine-HCI, pH 2.5. These affinity-purified antibodies were reacted with a nitrocellulose blot of a partially purified DNA polymerase I preparation separated by polyacrylamide gel electrophoresis (Figure 2). The unfractionated serum reacts with three polypeptides (145,000 daltons; 130,000 daltons; and 55,000 daltons), while the affinity-purified antibodies react only with the 145,000 dalton protein (Figure 2, lanes a and b). The 145,000 dalton polypeptide was shown to correspond to polymerase by purifying the catalytic core subunit of DNA polymerase I to homogeneity, as described in Experimental Procedures. Electrophoresis of the purified DNA polymerase I in polyacrylamide gels in the presence of SDS yields a single polypeptide of 145,000 daltons (see Experimental Procedures), which is shown to correspond to the catalytic subunit by analysis of DNA polymerase activity within the gel (Figure 2, lanes c and d). Therefore, the antibodies that react with the class l-encoded proteins also bind specifically to DNA polymerase I. Overproduction of DNA Polymerase Activity in S. cerevisiae An independent method to identify the recombinant phage containing the DNA polymerase I gene is to isolate genomic clones in a multicopy vector and assay for overproduction of polymerase activity in yeast. Representatives of the six major classes were used as probes to screen a yeast library carrying 10-20 kb inserts in the high copy number vector YEp24 (Carlson and Botstein, 1982). Two to three clones were isolated for each class, and these

Figure 3. RNA Blot Analysis Containing Cells

of pOly(A)

RNA Isolated

from

Plasmid-

Poly(A) RNA was isolated from YM214 containing either YEp24, pPOLl-1 or pPOLl-2, which are YEp24 plasmids with sequences homologous to class I clones. After electrophoresis and transfer to nitrocellulose, the RNA was hybridized with a nick-translated restriction fragment (the internal Sal I restriction fragment; see Figure 5). The arrow indicates a transcript of approximately 5.4 kb.

were then transformed into the haploid yeast strain YM214. Crude lysates of transformed yeast were assayed for DNA polymerase activity. The results for two YEp24 recombinant plasmids that contain sequences homologous to class I Agtll clones are shown in Table 1. The presence of either plasmid in the yeast cell results in a 4- to 5-fold increase in the specific activity of DNA polymerase above the control, YEp24. RNA blot analysis shows that each plasmid encodes a 5.4 kb, class l-homologous poly(A) RNA, which is overproduced more than lo-fold in yeast cells containing these plasmids (Figure 3). This message is sufficiently large to encode a 210 kd protein. Synthesis of Yeast DNA Polymerase I in E. coli Further evidence that the isolated gene encodes DNA polymerase I, was obtained by inserting an intact gene into an E. coli expression vector and assaying extracts for yeast polymerase activity. E. coli polymerase activities and the yeast polymerase activity were distinguished using both specific inhibitors and the anti-yeast DNA polymerase I antibodies. All three E. coli DNA polymerases are inhibited by dideoxy-TTP, whereas the yeast polymer-

Cell 372

probe :

plosmid

mpl8

:

subclone

LYJ

mpl9

-7 i

-7

2 Q

i

(1

subclone * c-u ?

Figure

5. Restriction

Map of POL7 Clone

The restriction map was determined from pPOLl-1, which contains a 9 kb insert in the Barn HI restriction site of YEp24. The direction of transcription is indicated (the exact 5’and 3’ end points have not been determined). The portions of the gene contained within two of the class I A clones are shown, and the fusion to the lacZ gene is at the 5’ end. The Eco RlXho I restriction fragment that was subcloned into M13mp18 (mp18-l), M13mp19 (mp19-l), is indicated, and the DNA contained in oPOU-2 is also shown.

Table 2. DNA Polymerase Containing E. coli Cells* Figure

4. RNA Blot Analysis

Using

Strand-Specific

Probes

Poly(A) RNA was isolated from cells containing either YEp24 or pPOLi-1, electrophoresed, and transferred to nitrocellulose. Hybridization was carried out using either labeled M13mp18-1 (mp18 subclone) or M13mp19-1 (mp19 subclone) as probes. The M13mp19-1 probe hybridizes to the 5.4 kb RNA, whereas the M13mp18-1 probe only hybridizes to nucleic acid with a very low mobility. This nucleic acid is much larger than the 5.4 kb RNA identified in Figure 3 and was not further analyzed.

a8es are unaffected by its presence (Chang, 1977). Conversely, the yeast polymerases are inhibited by aphidicolin, and the E. coli polymerases are not (Chang, 1977). Through combinations of these two inhibitors, it is possible to distinguish between the two activities in an extract. Before cloning in E. coli, the direction of transcription was determined as described in Experimental Procedures. When the topoisomerase II gene was isolated from the Igtll library, some of the recombinant phage contained the inserted gene in the appropriate orientation to make a fusion protein, while other phage contained inserts in the opposite orientation (Goto and Wang, 1984). Thus, it cannot be assumed that the inserted DNA is transcribed and translated in the same direction as fi-galactosidase. However, from the results shown in Figure 4, it is deduced that the direction of transcription is from left to right, as shown in Figure 5, and that at least two of the lambda clones isolated in this study have inserts in the same orientation as /acZ, as shown in Figure 5. The yeast polymerase gene was inserted downstream from the phage T7 RNA polymerase promoter in the vector pT7-I (Tabor and Richardson, 1985; see Experimental Procedures). The recombinant plasmid, pPOLl-4, was used to transform a strain carrying T7 RNA polymerase cloned in a compatible plasmid. Induction of T7 RNA polymerase allows transcription to be initiated at the T7 promoter and to continue through the DNA polymerase gene (Tabor and Richardson, 1985). If the appropriate signals are available near the beginning of the DNA polymerase gene, translation will be initiated, and a polymerase protein will be made. Induction is carried out in the presence of rifampicin to inhibit new synthesis by the E. coli RNA

Activity

in Plasmid-

% DNA Polymerase Activity*

lnhibitort ddTTP

+ +

wh

o-Poll

pT7-1

POLI -4

+

100 22 24 89 93

288 164 20 87 94

+ +

* DNA polymerase activity determined for JMlOl containing plasmids pGPl-2 and T ddTTP, dideoxy-TTP; aph, aphidicolin; merase I antibody. $ 199% activity = 41 pmol total nucleotide

5 rg crude extract made from either pT7 or pPOLl-4. o-Poll, anti-yeast DNA polyincorporated

in 1 hr at 35OC.

polymerase. In our experiments, this treatment resulted in a lo-fold reduction of E. coli DNA polymerase activity relative to uninduced extracts (data not shown). Extracts of the plasmid containing cells were assayed for DNA polymerase (Table 2). The level of activity in pT7-1 cells is considered the 100% control value. In the absence of any inhibitors, the cells containing the DNA polymerase I gene (plasmid pPOLl-4) have 166% more polymerase activity than cells containing the parental vector pT7-1. The addition of dideoxy-TTP inhibits the E. coli polymerases to 22% of the uninhibited level (pT7-1 column), whereas the yeast polymerase is uninhibited, resulting in 164% of the activity remaining (pPOL1-4). When aphidicolin is included in the reaction mixture in addition to ddTTP extracts from pPOL1-4 cells are now inhibited to 20% of the control activity, whereas extracts from pT7-1 cells are not further inhibited. Aphidicolin alone does not inhibit pT7-1 extracts. In contrast, addition of either aphidicolin alone or anti-yeast DNA polymerase I antibody inhibits the pPOLl4 extracts to approximately 90% of the uninhibited pT7-1 extracts, indicating that the aphidicolin-sensitive polymerase activity is contributed by yeast DNA polymerase I and not by DNA polymerase II. These results demonstrate the presence of DNA polymerase I in cells containing the yeast gene. The data presented above demonstrate that the isolated gene encodes DNA polymerase I, and we therefore designate the gene POLl.

;;Ft

DNA Polymerase

Table

3. Tetrad

I Gene

Analysis

of the ura3-52hra3-52

Viable Spores Per Tetrad’

Number Observed

4 3 2 1 0

0 0 16 1 1

Diploid

(CIT3)

Genotype ura*

0 0

Transformed

with Plasmid

ura-

Terminal Phenotype Inviable Sparest

36 1

A B C D

of

pPOLl-1 Number Observed 33 2 1 4

Percent* 92 5 3

* Total number of tetrads = 20. T Terminal phenotype determined 6 hr after germination as follows: A, singly budded cell with nucleus located at the mother-bud phenotype A with additional small bud at junction between mother and daughter cell; C, one large cell with two or more abnormal ungerminated spore. * Percent of germinated spores.

Gene Disruption and Copy Number of the POL7 Gene The copy number of the POLl gene was determined by Southern analysis. In an Eco RI digest of DNA from the haploid strain YM214, the expected four bands were detected that hybridized with the Sam HI fragment containing the POL7 gene (800 bp, 3000 bp, 3700 bp, and 8000 bp; data not shown; see Figure 5). No further bands were detected, consistent with the POL7 gene being present in yeast genome in a single copy. To determine the role of the DNA polymerase I gene in vivo, a mutant deficient in DNA polymerase I was constructed. To accomplish this, the gene was disrupted by homologous integration of a nonreplicative plasmid containing a DNA fragment solely within the coding region of the POD gene. A diploid strain was transformed with pBR322 containing a small fragment of the polymerase I gene and URA3 (Figure 5). Stable Ura+ transformants were isolated and the site of integration of the transforming DNA was determined by Southern analysis (data not shown). One transformant that was integrated at the POL7 gene, was then sporulated, and the resultant tetrads were analyzed (Table 3). Eighteen out of twenty tetrads segregated 2 viable; 2 inviable spores, where all of the viable spores yielded Ura- colonies. Since the URA3 gene is physically linked to the disrupted polymerase gene, these results indicate that the lethal mutation is genetically linked to the disrupted gene. Therefore, DNA polymerase I is a single copy, essential gene. Examination of the inviable germinated spores revealed a terminal phenotype typical of cell division cycle mutations that have a defect in replication, a large, singly budded cell with the chromosomes located in the junction between the mother and daughter cell (Hartwell, 1971; Figure 8). The data in Table 3 indicate that 82% of all spores examined and 92% of all germinated spores have this terminal phenotype. This morphology is similar to that for cdc8 (thymidylate kinase) mutants held at the nonpermissive temperature (data not shown; Culotti and Hartwell, 1971). Discussion The gene for yeast DNA polymerase I has been isolated using antibodies to screen a genomic Agtll library. The identity of the polymerase I gene was confirmed by three methods. First, we demonstrated immunological cross-

junction; B, buds; and 0,

reaction of affinity-purified antibodies to peptides synthesized from Agtll with DNA polymerase. Then we showed overproduction of polymerase activity in yeast when the gene was contained on a multicopy vector. Finally, we were able to observe synthesis of yeast DNA polymerase I in E. coli cells containing the appropriate clone. The DNA polymerase I gene is given the designation POL7 and is the first cloned eukaryotic DNA polymerase gene. Verification of the desired gene among the initial clones identified in immunological screens is often difficult. The methods used in this work are worth emphasizing. First, for a gene of unusually large size, such as DNA polymerase I, the frequency with which the gene is isolated in a genomic library screen and RNA blot analysis can suggest which clones to study further. The finding that the largest class, class I, contains 42 members and encodes a large 5.4 kb RNA led to further analysis of these clones. A more general and novel technique was employed to yield direct evidence that class I clones encoded yeast DNA polymerase. Protein lysates of Igtll clones were prepared directly on filters, and these protein-coated filters were used to affinity-purify antibodies that were used for immunoblots (Snyder and Davis, 1985). This is a useful approach for rapidly determining which clones encode antigens related to the protein of interest and eliminates obvious false positives. It was particularly useful in this case because the DNA polymerase I antiserum had been made against an impure preparation of the enzyme. An independent technique was also employed. The DNA polymerase was cloned into the pT7-1 expression vector of Tabor and Richardson (1985), which allows expression in an E. coli host carrying the cloned T7 RNA polymerase gene and allows detection of the yeast gene product in the bacterium. In the current work, expression of the yeast gene was monitored by enzymatic assay. One of the special features of the T7 expression system (as opposed to other bacterial expression vectors) that made this possible is the high levels of expression of the foreign gene coupled with the reduced expression of the endogenous activities. It is not clear what signals are used for translation of the foreign gene or if genes from other organisms are expressed as easily. This expression system works well with other yeast genes, as demonstrated by the expression of the yeast SSB-1 gene (single-stranded binding protein) in E. coli (Jong and Campbell, unpublished results). The role of DNA polymerase I versus polymerase II in

Cell 374

Figure

6. DAPI-Stained

Tetrads were germinated DNA-specific fluorescent

Cells

Containing

the Disrupted

DNA

Polymerase

I Gene

after 1 week on sporulation plates and were fixed with methanokacetic acid (3:l). The fixed cells were stained with the probe DAPI (1 &ml) and were observed under a Zeiss standard microscope for epifluorescence. Bar = 5 rm.

replication and DNA repair is still unknown. To determine whether they have overlapping functions and whether polymerase I is encoded on a single copy gene, we have carried out genomic Southern hybridization experiments, have disrupted the POL7 gene, and have analyzed the ef-

fects by Southern blot and tetrad analysis. These experiments revealed that the gene is present as a single copy and that disrupting the POL7 gene is lethal to the cell, indieating that it is an essential gene. From this we deduce that DNA polymerase II cannot replace polymerase I,

Yeast DNA Polymerase I Gene 375

though it may also be essential. It should now be possible to isolate conditional lethal and aphidicolin-resistant mutants in the POLl gene through mutagenesis of the cloned gene. These mutants would be useful for analysis of in vitro replication systems, the isolation of extragenic SUPpressors to identify interacting proteins, and analysis of the other DNA polymerases in yeast. Hartwell and coworkers found that when mutants defective in DNA replication were incubated at the nonpermissive temperature, they were unable to proceed past medial nuclear division (Culotti and Hartwell, 1971). The mother cell and bud continue to grow, but the nucleus remains in the junction between the cells. This terminal phenotype arises because the unreplicated chromosomes cannot be segregated into the two cells, but migration to the isthmus is not hindered. There are 14 mutants that terminate at or near this point in the cell cycle, including cdc8 (thymidylate kinase), cdc9 (DNA ligase), and cdc27 (thymidylate synthetase). The germinated spores containing the disrupted POL7 gene were examined and were found to give this same terminal phenotype (Figure 6). A gene disruption can be thought of as a mutation affecting the synthesis of the gene product but not affecting any protein or mRNA already existing in the spore. When spores were dissected after only 3 days on sporulation plates, many of the cells with the disrupted POL7 gene were able to complete l-3 generations before terminating at medial nuclear division (data not shown). This implies that either the protein or the mRNA is stored in spores for several days. However, after 4-5 days on sporulation plates, the cells have lost the ability to complete even one round of replication and terminate in the first cell cycle. The observation that cells with the disrupted gene do terminate at medial nuclear division is consistent with the hypothesis that DNA polymerase I is essential for DNA replication. Biochemical analysis of DNA polymerase I has been hindered by its extreme sensitivity to proteolysis. Catalytically active polypeptides ranging from 75,000 daltons to 160,000 daltons have been isolated (Badaracco et al., 1963; Plevani et al., 1965) but the validity of the biochemical studies of these partially degraded proteins is questionable. Poly(A) RNA synthesized from the POL7 gene is approximately 5.4 kb, thus it has the potential to encode a protein of 210,000 daltons. Determining the DNA sequence of the POL7 gene should establish an accurate molecular weight of the newly synthesized protein. However, this will not establish whether there is proteolytic processing that is important for polymerase function. The possibility that the mature protein participating in replication is smaller than the primary translation product is being investigated using the isolated gene to construct deletions followed by gene replacement. In addition, overproduction of DNA polymerase in both yeast and E. coli should assist the isolation of substantial amounts of the undegraded protein. Experimental Procedures Strains

and Media

E. coli strain

Y1090

(AIacU169

A/on araD139

strA supf

trpC22::TnlO

pMC9; Young and Davis, 1984) was used for the Agtll screening; JMlOl (F’trsD36 /acP Z,,,,5pmAE/A(/ac-pro) supE thi; Messing et al., 1981) was the host strain for Ml3 phage and for the synthesis of yeast DNA polymerase in E. coli. S. cerevisiae strain YM214 @his38200 @2-807 ede2-767 ura3-52; Mark Johnston) was used to assay overproduction of DNA polymerase I, and the diploid strain CIT3 (YM214xYM259: al~his3A200lhis3a200 lys2-801/+ tyrl/+ ade2-lOl/ade2-101 Ura352/ura3-52) was used for the gene disruption experiment. PEP4D (alahisl/+ trpl/+ prcl-126/prcl-126pep4-3@ep4-3prbl-1122/prbl-1122 canVcan7; (Jones, 1977) was used to isolate DNA polymerase I. Bacterial media were made as described by Miller (1972), and yeast media (YPD-yeast extract, peptone, dextrose, or SD-synthetic minimal) were described by Sherman et al. (1979).

Reagents and Enzymes DAPI (4,6-diamidino-2-phenyl-indole) was obtained from Accurate Chemical & Scientific Corp. [&*P]dTTP (3000 Cilmmol) was obtained from Amersham; ‘H-methyl-dTTP (45 Cilmmol) was from ICN and ?protein A (>30 mCi/mg) was obtained from Amersham. Restriction enzymes and E. coli DNA polymerase I were obtained from either Bethesda Research Laboratories or New England Biolabs.

Plasmids

and Recombinant

Libraries

pPOLI-3 was constructed by cloning the Eco RI-Xho I restriction frag ment from pPOLi-1 (Figure 5) into the Eco RI-Sal I restriction sites of pBR322. The Hind Ill fragment containing the URA3 gene was isolated from YEp24 (Carlson and Botstein, 1982) and was inserted into the internal Hind Ill site (between the Eco RI and Xho I sites; Figure 5). The Eco RI-Xho I restriction fragment was also inserted into the Eco RI-Sal I restriction sites of M13mp18 and M13mp19 (mp18-1 and mp19-1). pPOLl-4 was constructed by inserting the 9 kb Barn HI restriction fragment from pPOLl-1 into the Barn HI restriction site of pT7-1 (Tabor and Richardson, 1985). Plasmid pT7-1 was kindly provided by U. S. Biochemicals. The orientation of the insert was determined by restriction mapping. Plasmid pGPl-2 carries phage T7 RNA polymerase and was the gift of Stan Tabor, Harvard Medical School. The Igtll library is described elsewhere (Snyder and Davis, unpublished), and the YEp24 library was a gift from D. Botstein (Massachusetts Institute of Technology, Cambridge, MA).

Preparation

of Antibody

Probe

Anti-E. coli antibodies were removed from antisera by pseudoscreening. A fresh overnight culture of Y1090 cells was grown on L broth, 0.2% maltose, and 50 pglml ampicillin. One-tenth of a milliliter of cells was infected with lo5 1gt11 vector and was plated on 150 mm L plates using 6.25 ml top agar. The plates were incubated for 3 hr at 42’C, and then each plate was overlayed with a nitrocellulose filter that had been previously soaked and dried in 10 mM IPTG. After 4 hr at 3pC, the filters were removed and soaked in TBS with 20% FCS (TBS = 150 mM NaCl and 50 mM pi.+HCI; FCS = fetal calf serum) for 45 min. Each filter was next treated with 12.5 ml of anti-DNA polymerase I serum (Chang, 1977) diluted I:100 in TBS with 20% FCS and incubated 1.5 hr at room temperature. The antiserum was saved, and the procedure was repeated twice. The Agtll L plates were reused once; after removing the first filter, the plate was overlayed with a second IPTGtreated filter and incubated an additional 2 hr at JPC.

lmmunoscreening with Anti-DNA

the Agtll Genomic Polymerase Antibody

DNA Library

Two million phage (1 x 10’ recombinants) were plated on Y1090 at a density of IO5 phage/l50 mm plate and were incubated as described above. In initial experiments, IPTG-treated filters were incubated on the plates for 2 hr. In subsequent experiments, filters were incubated for 8-9 hr, which increased signals 5 to lo-fold. Screenings were carried out in duplicate. After removing the filter, a second filter was overlaid on the plates and was incubated at 3pC for an additional 2 hr. The filters were treated at room temperature as follows (Young and Davis, 1984). They were first soaked for 45 min in TBS with 20% FCS, then in 12.5 ml of diluted antibody solution per large filter for l-2 hr. Filters were washed sequentially in TBS; TBS with 0.1% NP40; followed by TBS and then were treated with ~251-labeled protein A (> 30 mCi/mg) at a concentration of 1 &i in 10 ml TBS with 0.1% BSA per filter for

l-l.5 hr. Filters were washed in TBS, TBS with 0.1% NP40 twice, and again in TBS and were exposed to X-ray film. Positives that appeared on the duplicate filters were picked and rescreened twice to isolate individual clones.

Hybridizations Plaque Hybridizations Transfer of phage DNA onto nitrocellulose filters (Schleicher and Schull BAE5) was performed as described by Benton and Davis (1977) except that the phage were spotted on an overlay of T medium with 0.7% agarose on T plates. Hybridization and washing of the nitrocellulose replicas was done as described by Maniatis et al. (1982).

Colony

Hybridizations

Approximately lo’-10” cells were spread on L-agar plates containing 100 @/ml ampicillin (150 mm plates) and were incubated at 3pC. The resultant colonies were lifted onto nitrocellulose filters and were amplified on plates containing IO pglml chloramphenicol. Cell lysis and hybridization to the nitrocellulose filter was performed as described by Maniatis et al. (1982).

RNA Blot Analysis Poly(A) RNA was isolated from plasmid-containing cells grown on SD medium lacking uracil as described by Domdey et al. (1984). Five micrograms poly(A) RNA per lane was separated on 2.2 M formaldehyde 1% agarose gelsand transferred to nitrocellulose paper (Maniatis et al., 1982). Hybridizations were carried out at 42OC in the presence of 50% formamide, 0.8 M NaCI, 0.1 M PIPES, 0.01% sarcosyl, 5x Denhardts, 500 pglml denatured calf thymus DNA, and 10% dextran sulfate for 24-48 hr. Filters were washed 4 times at room temperature in 2x SSC, 0.05% sarcosyl, and 0.02% NaPPi and 4 times at 50°C in 0.2x SSC, 0.05% sarcosyl, and 0.01% NaPPi.

Probes DNA restriction fragments were isolated from low-melting-temperature agarose gels and nick-translated using (&*P]dTTP (3000 Cilmmol) the source of label (Maniatis et al., 1982).

as

Yeast Extracts YM214 containing the appropriate plasmid was grown on SD medium lacking uracil(30 ml) to an Asgo = l-2. After centrifugation, cells were resuspended in 1 ml of buffer A (50 mM Tris-HCI, pH 8.0; 50 mM NaCI; 1% DMSO; 10% glycerol; 5 mM p-mercaptoethanol; 10 mM EDNA; 2 mM benzamidine; and 1 mM PMSF). Lysis was achieved by vortexing the cells 8 times for 30 set in the presence of an equal volume of glass beads at 4%. The extract was transferred to an Eppendorf tube and centrifuged IO min.

E. coli Extracts JMIOI containing pGPl-2 (a pACYC derivative that contains both the T7 RNA polymerase gene under PL control and the cIaJ7 gene) and pT7-1 (control plasmid) or pPOLl-4 (DNA polymerase I gene downstream form the T7 promoter) was grown in L broth, 40 &ml kanamytin sulfate, and 100 pglml ampicillin (20 ml) to an ASoa = 0.8 at 3OOC. The T7 RNA polymerase was heat-induced by shifting the cultures to 42OC for 20 min and then 200 pg/ml rifampicin was added to inhibit E. coli RNA polymerase. Incubation was continued at 42OC for 8 min, followed by 1 hr at 3oOC. After centrifugation, cells were resuspended in 1 ml of buffer A and lysed by sonication. Debris was sedimented by centrifugation.

DNA Polymerase

Assays

DNA polymerase activity was assayed by incubating extracts with 50 mM ‘h-is-HCI (pH Z5), 2 mM /3-mercaptoethanol, 8 mM MgCI,, 50 mM KCI, 150 rglml activated salmon sperm DNA, 100 rg/ml bovine serum albumin, 100 PM dATP, dGTP: dCTR and 50 FM dTTP (3H-methyl-dTTP, 400 cpm/pmol) at 35OC for 20 min. When 100 PM dideoxy=TTP or IO pglml aphidicolin were added as inhibitors, the dTTP and dCTP concentrations were reduced to 5 PM (Table 2). One microliter of undiluted anti-yeast DNA polymerase I antibodies was added to the reaction mixture where indicated. Reactions were stopped by spotting 50 ~1 on Whatman DE-81 filter paper, and unincorporated nucleotides were removed by washing 5 times with 0.5 M Na,HPO,, once with H,O, and once with 95% ethanol.

Purlflcatlon

of DNA Polymerase

I

DNA polymerase I was purified from 500 g of frozen PEP4 cells according to the protocol of Badaracco et al. (1983) with the following modifications: buffers contained 10 mM EDTA, 2 mM benzamidine, and 1 mM PMSF to reduce proteolysis. The cells were lysed with glass beads using an lmpandex Dyno-Mill agitator and were centrifuged at 10,000 xg in a Sorvall GS3 rotor for 40 min. The Polymin P precipitate was extracted twice with buffer A plus 1.0 M NaCl and was precipitated with 50% ammonium sulfate. The dialyzed fractions were chromatographed on phosphocellulose (15.9 cm2 x 5 cm) as described previously (Badaracco et al., 1983). The active fractions obtained from the phosphocellulose column were precipitated with 50% ammonium sulfate, and the resuspended pellet was loaded on a heptyl-agarose column (23.8 cm’ x 2 cm). The column was washed with buffer B (25 mM potassium phosphate, pH 7.2; 200 mM KCI; 5 mM p-mercaptoethanol, IO mM EDTA, 2 mM benzamidine, 1 mM PMSF, 1% DMSO, and 10% glycerol) plus 35% ammonium sulfate. Polymerase activity was eluted with a gradient of 35%-O% ammonium sulfate in buffer B. The active fractions were concentrated, dialyzed, and loaded on a single-stranded DNA cellulose column (3 cm2 x 2 cm). Polymerase activity was eluted as described by Badaracco et al. (1983). After concentration, the active fractions were chromatographed through a Sephadex G-100 column (0.44 cm’ x 19 cm), and the polymerase activity was found to comigrate with a single polypeptide of 145,000, which was greater than 90% pure as estimated by polyacrylamide gel electrophoresis.

Affinity

Purification

of Polymerase

I Antibody

Proteins synthesized from class I clones were used to affinity-purify antibodies as described in Snyder and Davis (1985). The appropriate a clone was plated on strain YlO90 to yield 3 x l(r plaques per 90 cm plate. After 3 hr at 42OC, filters soaked in IO mM IPTG were applied and left overnight at 3PC. Filters were removed and treated with antibody, from which E. coli antibodies had been removed, exactly as described in the screening procedure. After washing away unbound antibody, the filter was placed in a 15 ml plastic tube. Bound antibody was eluted by adding 3.5 ml 0.2 M glycine-HCI (pH 2.5), and shaking for 2 min. The filter was removed, and the antibody solution was neutralized with 1.75 ml of 1.0 M potassium phosphate buffer (pH 9.0), containing 5% fetal calf serum. For protein blotting, the neutralized solution was diluted with 8 ml H20, 1 ml fetal calf serum, and 3 ml TBS (Burnette, 1981).

Direction

of Transcription

of the POLl

Gene

To determine the direction of transcription, an RNA blot ana!ysis was carried out using single-stranded DNA probes containing either strand of the polymerase gene. An Eco RlXho I restriction fragment was cloned into both M13mpl8 and Ml3mpl9 (Figure 5), and the isolated single-stranded DNA was labeled by priming was the Ml3 Hybridization Probe-Primer (New England Biolabs) and was labeled with [o-32P]dTTP as described by Hu and Messing (1982). The results of the RNA blot analysis using these probes is shown in Figure 4. The Ml3mpl9 probe hybridizes to the 5.4 kb mRNA, whereas the Ml3mpl8 probe does not. Two of the recombinant phage were analyzed and found to have the POLI gene in the same orientation as the laci’gene.

ELISA Assay

with Anti-DNA

Polymerase

Monoclonal

Antlbodles

Protein lysates were prepared from phage-infected cells by first preparing lysogens of individual positive clones on E. coli Y1089 (AIacU769 proA+ A/on araD739 strA hflA7 [chr:TnlO] pMC9; Young and Davis, 1984). Lysogens were prepared for three members of class I, two members each of class II, Ill, and VII, and one member each of class IV, V, and VI, all of which showed a strong reaction with the polyclonal antiDNA polymerase serum. Three nonreactive clones and Agtll vector were also tested. Lysogens were grown in 5 ml L broth to an A,00 = 0.25, induced by incubation at 44OC for 15 min, and IPTG was added to IO mM and incubated at 3PC for 45 min. Cells were collected by centrifugation, washed once with PBS (150 mM NaCI, 10 mM NaPO,, pH 7.4), resuspended in 150 ~1 of TBS, and frozen at -70°C to lyse the cells. Thirty microliters of each protein lysate was added to a microtiter well and absorbed for 2 hr at room temperature. The solution was removed, and the wells were treated at room temperature with -50 ~1 of PBS with 20% FCS for 45 min and rinsed with PBS. Four mouse

Yeast 377

DNA Polymerase

I Gene

monoclonal antibodies that react with DNA polymerase I (Plevani et al., 1984) were pooled and diluted at I:40 (per monoclonal antibody) in PBS with .I% BSA, and 30 ~1 was added to each well for 2 hr at room temperature. The wells were washed 6 times with double-distilled water. Thirty microliters of alkaline phosphatase linked to goat anti-mouse antibody (Boehringer Mannheim), diluted in PBS with .I% SSA was added for 1 hr at room temperature, and the wells were washed 6 more times with water. One hundred microliters of pnitrophenyl substrate and 10% diethanolamine was added for l-4 hr, and the reaction was stopped by adding 6 pI5M NaOH. Absorbance at 414 nm was read in a Titertech microtiter well spectrophotometer. The experiment was repeated three times with the mouse monoclonal antibodies and performed once using the polyclonal anti-polymerase serum.

We thank Stan Tabor and Charles Richardson for communicating results prior to publication. This work was supported by an NIH postdoctoral fellowship to Lianna M. Johnson, a Helen Hay Whitney fellowship to Michael P Snyder, NIH Grant #MG5-ROIGM-25506-07, American Cancer Society Grant #MV-1426, and March of Dimes Grant #l-794 to Judith L. Campbell, and an American Cancer Society Grant to Ronald W. Davis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. June

16, 1985; revised

August

7, 1985

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by

II genes: