Genetic and molecular analysis of DNA43 and DNA52: two new cell-cycle genes in Saccharomyces cerevisiae.

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.

Reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex in vitro. The 86-kDa subunit facilitates but is not required for complex formation.

The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex. The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S. The calculated molecular weight of the reconstituted complex is 312,000. We infer that the stoichiometry of the complex is one of each subunit per complex. The complex has a prolate ellipsoid shape with an axial ratio of approximately 16. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody. While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits). The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact. However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity. The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit. The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.

Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities.

The yeast Saccharomyces cerevisiae catalytic DNA polymerase I 180-kDa subunit and the tightly associated 86-kDa polypeptide have been purified using immunoaffinity chromatography, permitting further characterization of the DNA polymerase activity of the DNA primase-DNA polymerase protein complex. The subunits were purified to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. When the individual subunits were recombined in vitro a p86p180 physical complex formed spontaneously, as judged by immunoprecipitation of 180-kDa polypeptide and DNA polymerase activity with the anti-86-kDa monoclonal antibody. The 86-kDa subunit stabilized the DNA polymerase activity of the 180-kDa catalytic subunit at 30 degrees C, the physiological temperature. The apparent DNA polymerase processivity of 50-60 nucleotides on poly(dA).oligo(dT)12 or poly(dT).oligo(A)8-12 template-primer was not affected by the presence of the 86-kDa subunit but was reduced by increased Mg2+ concentration. The Km of the catalytic 180-kDa subunit for dATP or DNA primer terminus was unaffected by the presence of the 86-kDa subunit. The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis that had been observed previously in the DNA primase-DNA polymerase protein complex. The 180-kDa subunit possessed a 3'----5'-exonuclease activity that catalyzed degradation of polynucleotides, but degradation of oligonucleotide substrates of chain lengths up to 50 was not detected. This exonuclease activity was unaffected by the presence of the 86-kDa subunit. Despite the striking physical similarity of the DNA primase-DNA polymerase protein complex in all eukaryotes examined, the data presented here indicate differences in the enzymatic properties detected in preparations of the DNA polymerase subunits isolated from S. cerevisiae as compared with the properties of preparations from Drosophila cells. In particular, the 3'----5'-exonuclease activity associated with the yeast catalytic DNA polymerase subunit was not masked by the 86-kDa subunit.

DNA primase isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis.

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.

The CDC8 transcript is cell cycle regulated in yeast and is expressed coordinately with CDC9 and CDC21 at a point preceding histone transcription.

Using cultures synchronized by elutriator size selection or a feed-starve protocol, we have shown that the CDC8 gene is periodically expressed in the Saccharomyces cerevisiae cell cycle. The transcript level increases some 30-fold in late G1, reaching a peak at approximately the G1/S phase boundary. The timing of this event was compared with those of CDC9 and CDC21, which are already known to be periodically transcribed, and all three genes were found to be expressed at the same time in the cell cycle. In contrast, the histone H2A gene appeared to be expressed distinctly later in the cell cycle than these three genes and this was further investigated by examining expression of all four genes in a cdc4 mutant, held at the restrictive temperature. CDC8, CDC9, and CDC21 were once again expressed together and a complete fluctuation in levels occurred, whereas the histone gene was not expressed, presumably because the cdc4 block point precedes the point of histone expression. The three CDC genes may therefore be coordinately controlled, while the histone gene is regulated separately.

Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis.

The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.

A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae.

Biochemical fractionation of the yeast Saccharomyces cerevisiae has revealed a novel DNA primase activity that copurifies with the major DNA polymerase activity. In the presence of RNA precursors and single-stranded DNA (poly(dT), M13), the DNA primase synthesizes discrete length oligoribonucleotides (apparent length, 8-12 nucleotides) as well as longer RNA chains that appear to be multiples of a modal length of 11-12 nucleotides. When DNA precursors are also present, the oligoribonucleotides are utilized by the accompanying DNA polymerase as primers for DNA synthesis. Copurification of these two enzymatic activities suggests their association in a physical complex which may function in the synthesis of Okazaki fragments at chromosomal replication forks.

Saccharomyces cerevisiae CDC8 gene and its product.

The product of the Saccharomyces cerevisiae CDC8 gene is essential for normal cellular DNA replication; the determination of the structure of the gene and the identification of its product would facilitate the examination of its role in this process. We have cloned a 1,000-base-pair fragment of the S. cerevisiae genome carrying the functional gene. The nucleotide sequence includes one long open reading frame; it is flanked by sequences typical of other S. cerevisiae genes. This sequence predicts a polypeptide chain product of 216 amino acids with a molecular weight of 24,600. A polyadenylated RNA transcript of this sequence was identified by hybridization; in vitro translation of RNA samples enriched for this transcript produced a specific polypeptide chain of apparent molecular weight between 24,000 and 25,000. Thus the reading frame identified represents the authentic CDC8 gene, and the amino acid sequence of its product has been deduced. Our observations differ from two previous reports of the identification of the putative CDC8 protein based upon in vitro complementation assays.

Use of plasmid-mediated resistance to the antibiotic G418 for the rapid screening of a yeast mutant library.

Both haploid and diploid yeast cells are sensitive to the antibiotic G418. The former develop spontaneous resistance to G418 at a frequency of approximately 2 X 10(-6), however, the frequency of resistance in diploids is less than 1 X 10(-9) and was undetectable in these experiments. Mating of spontaneously resistant haploid cells to G418-sensitive strains yields sensitive diploids, indicating that spontaneous resistance to the antibiotic is a recessive trait. Both haploid and diploid cells can be efficiently transformed to G418 resistance by a plasmid carrying the Escherichia coli kanamycin resistance (kanr) marker. The ability to select for cells transformed with plasmids containing the kanr gene has facilitated the screening of a large series of temperature sensitive yeast mutants to determine whether any of them are allelic to RAD3.

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I.

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication.

We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 11000 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictive temperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.

Initiation of DNA synthesis on the isolated strands of bacteriophage f1 replicative-form DNA.

Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage f1 replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on f1 viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of f1 viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on f1 complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the f1 replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for f1 DNA replication in vivo proposed by Horiuchi and Zinder.

Replication of bacteriophage phiK duplex replicative-form DNA in dnaB and dnaC mutants of Escherichia coli.

We have directly tested the effects of host cell DNA synthesis mutations on bacteriophage phiK replicative-form (RF) DNA replication in vivo. We observed that phiK RF DNA replication continued at normal rates in both dnaB and dnaC mutant hosts under conditions in which the activities of the dnaB and dnaC gene products were shown to be markedly reduced. This suggests that these two host proteins are not essential for normal phiK RF DNA replication. In control experiments we observed markedly reduced rates of phiK RF DNA replication in temperature-sensitive dnaG and dnaE host mutants, indicating that the products of these genes are essential. Thus, the mechanism of DNA chain initiation in vivo on the duplex RF DNA templates of isometric phages such as phiK apparently is different from that on the similar templates of isometric phages such as phiX174. The implications of this difference are discussed in the text.

Isolation of circular viral and complementary strand DNA from bacteriophage f1 duplex replicative-form DNA.

A general method has been developed for the large scale isolation of intact, circular, single-stranded DNA molecules of each strand from supercoiled duplex DNA. The method involves the conversion of the supercoiled duplex DNA to singly nicked, relaxed duplex DNA; denaturation of the duplex DNA; separation of circular DNA molecules from linear DNA molecules; and separation of circular plus and minus strands. All separations involve zone sedimentation. No isopycnic gradient centrifugation is required. The last step in the purification, the separation of plus and minus strands, can be easily adapted for small scale analytical measurements of the amounts of plus and minus strand DNA.

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.