Deoxyribonucleic Acid Synthesis in

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1 JOURNAL OF BACTERIOLOGY, Sept. 973, p Vol. 5. No. 3 Copyright 973 American Society for Microbiology Printed in U.S.A. Three Additional Genes Required for Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae LELAND H. HARTWELL Department of Genetics, University of Washington, Seattle, Washington 9895 Received for publication April 973 Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc, 7, and 8) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and ) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 3 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature. An extensive search for temperature sensitive cell division cycle (cdc) mutants of the yeast Saccharomyces cerevisiae revealed 5 cdc mutants. Genetic studies have shown that these mutations define 3 genes that control various steps in the cell division cycle of S. cerevisiae (L. H. Hartwell et al., manuscript in press). Evidence was present that the product of gene cdc was required for the initiation of deoxyribonucleic acid (DNA) synthesis, and the product of gene cdc 8 was required for DNA replication (7). This communication reports a more extensive analysis of the mutant collection in an attempt to find additional members that are defective in DNA synthesis. Evidence will be presented that thee additional genes also function in DNA synthesis, two in the initiation process, and a third in replication. MATERIALS AND METHODS Strains and media. All of the mutants were derived from the parent strain, A36A a ade I ade ura I tyr I his 7 IYs gal I (L. H. Hartwell et al., manuscript in press). All are isogenic with A36A, except for strains 35-- a, a, 6--3 a, and a which were derived from mutants 58, 39, 76, and 5 by outcrosses, since the latter four strains were found to contain more than one mutation. The experiments were carried out in synthetic media with the addition of.%7 yeast extract. Analytical techniques. Techniques for the analyses of radioactivity incorporated into DNA and ribonucleic acid (RNA) and for the staining of cells with Giemsa were described previously (6). Cells were scored as being unbudded, having small buds or large buds by visual examination in a phase-contrast microscope. Small buds are delined as less than half the diameter of the parent cell, and large buds as greater than half. Since the buds are not measured, some cells are ambiguous, having approximately half the diameter, and these are arbitrarily assigned to the small or large bud class. This ambiguity does not introduce a serious error since the progression of bud sizes is used as a qualitative indication of synchrony and not a quantitative measure. RESULTS Kinetics of DNA synthesis in asynchronous cultures. Mutants were examined for the kinetics of DNA synthesis after a shift from the permissive to the restrictive temperature. The pattern observed was compared with the kinetics of DNA synthesis in a parallel culture at the permissive temperature and in another culture at the permissive temperature to which cycloheximide was added. Cycloheximide does not inhibit on-going replication in yeast, but does inhibit the initiation of new rounds of DNA synthesis (L. M. Hereford and L. H. Hartwell, manuscript in press). The cultures were prelabeled for min prior to the addition of cy- 96(6

2 VOL. 5, 973 cloheximide or prior to the shift to the restrictive temperature to equilibrate deoxynucleotide pools. A defect in DNA replication would be expected to evidence itself by an immediate reduction in the rate of DNA synthesis after a shift to the restrictive temperature. A defect in the initiation of DNA synthesis should result in a pattern of DNA synthesis at the restrictive temperature that closely parallels the pattern of DNA synthesis seen at the permissive temperature after the addition of cycloheximide. The patterns of DNA synthesis for the parent strain, A36A, and for three mutant strains are illustrated in Fig.. The effect of the addition of cycloheximide at 3 C is similar for the parent strain and the three mutants. The initial rate of DNA synthesis was not immediately affected by the additibn of cycloheximide, but the rate of synthesis progressively decreased as a function of time after the addition. After h in cycloheximide, a slow but measureable rate of DNA DNA SYNTHESIS GENES IN S. CEREVISIAE 967 synthesis continued, and this synthesis is probably accounted for by mitochondrial DNA replication (5). The rate of DNA synthesis in the culture of the parent strain A36A at 36 C decreased transiently and then increased rapidly. The kinetics suggest a transient inhibition of the initiation of DNA synthesis in the parent strain after a shift from 3 to 36 C that lasts for about 5 min. Mutant strain 8 exhibits a pattern of DNA synthesis at 36 C very similar to that of the parent strain A36A for the first 3 h. After this time, the rate of synthesis in the mutant culture at 36 C is about (withih a factor of two) that shown). The amount of radioactivity that is incorporated by the mutant 8 culture after h at 36 C is about (with a factor or two) that expected if all cells synthesize a round of DNA before cell cycle arrest. To make this calculation, one must know the doubling time of the culture at the permissive temperature, the 8 I 3 a HOURS FIG.. The parental strain A36A (panel A) and the mutants 8 (cdc 6-; panel B), (cdc 8-, panel C) and 6--3 (cdc -; panel D) were grown in synthetic medium containing.% yeast extract at 3 C. T7he cells were centrifuged and resuspended in the same medium containing uci of "C-uracil per ml (specific activity, pci/. jg). The cells were incubated for min at 3 C to equilibrate the nucleotide pools and then divided into three samples ( time). One sample was shifted to 36 C (or 38 C for 85-3-) (A); a second received pg of cycloheximide per ml and was incubated at 3 C (U); and the third was left at 3 C without inhibitor as a control (). Samples were removed at various times and analyzed for radioactivity in DNA.

3 968 HARTWELL amount of radioactivity incorporated at the permissive temperature in one doubling time, and the amount of residual cell division that the mutant culture exhibits after a shift to the restrictive temperature. Mutant strain exhibits a pattern of DNA synthesis at the restrictive temperature that closely parallels that found at the permissive temperature after the addition of cycloheximide. This result suggests that mutant is defective in initiation of DNA synthesis. After h at the restrictive temperature, the rate of DNA synthesis is slightly greater than that in the cycloheximide-treated culture. We attribute this to "leakiness" of the defect in mutant The experiments with this strain are carried out at 38 C to minimize this leakiness. Mutant strain 6--3 exhibits an immediate cessation of DNA synthesis after a shift from 3 to 36 C as would be expected for a defect in DNA replication. Most of the mutant strains that were examined by this protocol fit clearly into one of the three categories as listed in Table. The mutants classified as having a defect in initiation exhibit patterns of synthesis at 36 C similar to that of mutant 85-3-, mutants classified as being defective in replication exhibit patterns similar to that of mutant 6--3, and mutants classified as having no defect exhibit patterns similar to that of mutant 8. From these initial studies, mutations in three genes appeared to cause a defect in the initiation of DNA synthesis (genes cdc, 7, and 8) and mutations in two genes (cdc 8 and ) cause defects in DNA replication. Previous work documented the conclusion that cdc mutants are defective in initiation of DNA synthesis and that cdc 8 mutants are defective in DNA replication (7). Another allele of cdc 7 (7- strain D) had been examined for DNA synthesis previously (), and it was noted that the mutant synthesized less than a complete round of DNA synthesis before cell cycle arrest. RNA synthesis in the DNA mutants. All of the mutants examined in this study are defined as cell division cycle mutants because the cells from an asynchronous population growing at the permissive temperature accumulate with a uniform morphology after a period of incubation at the restrictive temperature (8). Mutants of yeast defective in total RNA synthesis or in ribosomal RNA synthesis retain an asynchronous distribution of cellular morphologies after incubation at the restrictive temperature (unpublished observations). Thus, it was considered unlikely that the three newly discovered mutant strains defective in DNA synthesis J. BACTERIOL. (85-3-, cdc 8-; 8, cdc 7-; and 6--3, cdc -) were merely defective for total nucleic acid synthesis. Nevertheless, a comparison was made of the ability of these strains to synthesize RNA and DNA at the restrictive temperature. Mutant cultures growing at the permissive temperature were divided into two samples. One sample was left at the permissive temperature, and the other was shifted to the restrictive temperature. "C-uracil was added to both cultures at the time of the shift to label both RNA and DNA (all of the strains are uracil auxotrophs). The amount of radioactivity incorporated into RNA or DNA over a 3-h period at the permissive and at the restrictive temperature was determined and the ratio calculated (Table ). The amount of radioactivity incorporated into RNA at the restrictive temperature was about the same as that at the permissive temperature for the parent strain and the mutants. The amount of radioactivity incorporated into DNA at the restrictive temperature was slightly greater than that at the permissive temperature for the parent strain and less than % of that at the permissive temperature for each of the mutant strains. Therefore, the lesion in each of these mutants is specific for DNA synthesis. DNA synthesis in synchronous cultures. DNA synthesis was examined in synchronous cultures of each of the three newly discovered mutant strains defective in this process, (cdc 8-), 8 (cdc 7-), and 6--3 (cdc -). The first set of experiments were undertaken merely to verify the fact that these mutants were defective in some stage, either in initiation or replication, of DNA synthesis. These mutant strains are all haploids of a mating type, and could, therefore, be synchronized with a factor. The a factor is a substance produced by cells of a mating type that arrests cells of a mating type at a point in the cell cycle prior to the initiation of DNA synthesis and prior to bud emergence (). This substance is thought to play a role in the synchronization of the haploid cell cycles prior to cell fusion during zygote formation. The mutant strains were pretreated for about one cell cycle at the permissive temperature with a factor, and at the end of this time, most of the cells had accumulated as unbudded cells arrested prior to initiation of DNA synthesis. The a factor was then removed by filtration, and the cells were resuspended, half at the permissive temperature, and half at the restrictive temperature. The progress of the cells through the cell cycle was monitored morphologically by scoring the fraction of unbudded cells, the fraction of cells with

4 VOL. 5, 973 DNA SYNTHESIS GENES IN S. CEREVISIAE 969 TABLE. DNA synthesis kinetics of asynchronous cultures of cdc mutants cdc Gene f Allele Strain J Type of response TABLE E Replication defect Replication defect Replication defect Replication defect small buds (buds less than half the diameter of the parent cell), and the fraction of cells with large buds (buds larger than half the diameter of the parent cell). The results of these experiments are displayed in Fig. (85-3-, cdc 8-), in Fig. 3 (8, cdc 7-), and in Fig. (6--3, cdc -). A control experiment with the parent strain is shown in Fig. 5. The synchronous nature of these cultures is evidenced at the permissive temperature by the succession of morphological cell types from unbudded cells to cells with small buds to cells with large buds. In the culture of the parent strain, A36A, at the restrictive temperature both DNA synthesis and bud emergence are delayed relative to the culture at the permissive temperature, but otherwise the two cycles are similar. It is clear that all three mutant strains are deficient in DNA synthesis at the restrictive temperature, although they exhibit a low level of synthesis after prolonged incubation. This synthesis may be due to leakiness of nuclear DNA synthesis, or may be due to mitochondrial DNA synthesis. All three mutant strains undergo a normal cell cycle at the permissive temperature as evidenced by the budding pattern and DNA synthesis. The cells of mutant strain (cdc 8-) do not bud at the restrictive temperature, but continue to enlarge and elongate becoming "shmoo" shape reminiscent of cells that have been inhibited for prolonged periods of time with a factor (Fig. ; L. H. Hartwell et al., manuscript in press). This morphological appearance of cells of strain at the restrictive temperature is not dependent upon prior treatment with a factor, since the cells from asynchronous cultures incubated at the restrictive temperature assume the same appearance as, for that matter, do cells of a mating type that carry this mutation. Cells of strain 8 (cdc 7-) bud at the restrictive temperature, and the bud grows in size so that the cells terminate development with a single large bud. The nucleus migrates into the isthmus between the bud and the RNA and DNA synthesis at the permissive and restrictive temperatures RNA DNA Mutant gene Strain-T 3 C 36 C ratio 36: 3 3 C 36 C ratio 36: 3 A36A 9,8 96,. 5,6,6.3 cdc 7-8 7, 8,8.5,,3.9 cdc , 8,9.93,,3a.3 cdc ,3 67,8.67 3,7 8.5 a Performed at 38 C.

5 97 HARTWELL J. BACTERIOL. H z 8 C) c 6 ILI Nf "- U 5 3 Temperature shifts during the DNA synthetic period. The previous data obtained with synchronous cultures of the mutant strains clearly demonstrated that all three strains are defective in DNA synthesis. The experiments with synchronous cultures do not distinguish between a defect in processes required for the initiation of DNA synthesis or in processes required for DNA replication. A distinction can be made between these two possibilities by performing the shift from the permissive to the restrictive temperature after the DNA synthetic period has begun, but before it has been completed. If a mutant is defective in the initiation of DNA synthesis, then cells that have begun DNA synthesis at the permissive temperature should be able to complete DNA synthesis after a shift to the restrictive temperature, whereas if the mutant is defective in DNA replication, then a shift to the restrictive temperature at any H 8 z LiJ r 6 Lii 3 H U R S FIG.. DNA synthesis in a partially synchronous culture of mutant (cdc 8-) at the permissive and restrictive temperatures. Cells growing at 3 C were adjusted to a density of 5 x 6 cells/ml in ml of medium and. ml of a concentrated solution of a factor was added. The cells were shaken vigorously for h at 3 C, after which they were filtered and resuspended in 5 ml of fresh medium containing Ci of "C-uracil per ml (specific activity, 5 mci/mmole). The culture was divided in two parts; one was shifted to 38 C; and the other was left at 3 C. Samples were removed at various times and assayed for the amount of radioactivity incorporated into DNA (center panel). Samples were also removed and scored for the percentage of cells without buds (-), with small buds (-), and with large buds (A). 8 z LiJ cc- 6 N, a. 5 3 ~ ow Hz 8 z LI 6 cr- L) 6 Lii BUDDING 3 DNA- 3/ BUDDING 36 parent cell and assumes a morphology characteristic of a dividing nucleus (L. H. Hartwell et al., manuscript in press). Cells of strain 6--3 (cdc -) also undergo bud emergence and bud enlargement at the restrictive temperature, and the nucleus also migrates into the isthmus between the parent cell and bud (L. H. Hartwell et al., manuscript in press) H U R S FIG. 3. DNA synthesis in a partially synchronous culture of mutant 8 (cdc 7-) at the permissive and restrictive temperatures. Protocol is the same as Fig. except that the restrictive temperature was 36 C.

6 VOL. 5, 973 DNA SYNTHESIS GENES IN S. CEREVISIAE 97 shown that the execution point for a mutant strain defective in cdc (an initiation defect) o BUDDING 3 occurs prior to bud emergence at about the time that DNA synthesis begins, and a mutant strain >_8 X defective in cdc 8 (a replication defect) has an 6\ execution point considerably after bud emerw <-\ gence coinciding approximately with the end of the DNA synthetic interval. The execution points for mutant strains bearing the cdc 8- - mutation or the cdc 7- mutation are at.6 - and.3 fractions of a cell cycle, respectively, and are therefore at or prior to bud emergence which occurs at about.3 fractions of a cell 5 - cycle in the haploid cycle (L. H. Hartwell et al., / manuscript in press). The execution point for a 3 S3? ~ strain bearing the cdc - mutation was shown to be at.63 fractions of the cell cycle, consideri ably after bud emergence. Thus, these results in themselves strongly suggest that mutant strains 36 BUDDING 3 o L 9 * * ~~~~~~BUDDING 36 8 w HH8 w z a w a. - 3 FIG.. DNA synthesis in a partially synchronous L- culture of mutant 6--3 (cdc -) at the permissive 3- and restrictive temperatures. Protocol is the same as Fig., except that the restrictive temperature was 3 36 C. MO 36 DNA time during the DNA synthetic period should a A inhibit further synthesis. Actually, the requisite information is already BUDDING 38 available for these mutant strains since all of the cdc mutants have been characterized with H 8 respect to their execution points (L. H. Hartwell et al., manuscript in press). The executiont point c - 6 is defined as that time in the cell cycle at the. O - permissive temperature that the cell acquires the capacity to complete the present cell cycle - and arrest in the second cell cycle after a shift to the restrictive temperature. An initiation mu- tant should have an execution point that coin- 3 cides with the time of initiation of DNA synthe- H U R S sis, and a replication mutant should have an FIG. 5. DNA synthesis in a partially synchronous execution point that coincides with the end of culture of the parent strain A36A. Protocol is the the DNA synthetic period. Previous results have same as Fig..

7 97 HARTWELL J. BACTERIOL (cdc 8-) and 8 (cdc 7-) are defective in processes required specifically for initiation of DNA synthesis, whereas mutant strain 6--3 (cdc -) is temperature sensitive throughout the interval of DNA replication. The DNA synthesis patterns from asynchronous cultures (Fig. ) suggested the same conclusions. Nevertheless, we sought to verify these conclusions by examining the time course of DNA synthesis after a shift from the permissive to the restrictive temperature during the DNA synthetic interval. Partially synchronized cultures were prepared by arresting cells with a factor as before. The a factor was removed by filtration, and the cells were resuspended at the permissive temperature. At a time when DNA synthesis had begun in the culture but before extensive synthesis had occurred, samples were removed and shifted to the restrictive temperature or left at the permissive temperature with the addition of cycloheximide. It is assumed that the time course of synthesis in the culture receiving cycloheximide represents the completion of DNA synthesis in those cells that had initiated DNA synthesis at the time of inhibitor addition (L. M. Hereford and L. H. Hartwell, manuscript in press). The time course of DNA synthesis at the restrictive temperature closely parallels that in the cycloheximide culture for mutants (cdc 8-; Fig. 6) and 8 (cdc 7-; Fig. 7). We conclude, therefore, that the products of genes cdc 8 and cdc 7 are required specifically for steps involved in the initiation of DNA synthesis but not in the completion of replication once initiation has been successfully undertaken. DNA synthesis is arrested very rapidly in the culture of mutant 6--3 (cdc -) shifted to the restrictive temperature but not in the culture to which cycloheximide was added (Fig. 8). We conclude, therefore, that the product of gene cdc is required for DNA replication. DISCUSSION These results bring to five the number of genes thought to play a role in DNA synthesis in yeast. Three of the genes (cdc, 7, and 8) appear to function in the initiation of DNA synthesis, and two (cdc 8 and ) appear to function in DNA replication. Although this is the simplest interpretation of the data that has been presented, it is important to note that the distinction between a defect in initiation of DNA synthesis and one in DNA replication is based entirely upon the effects of temperature shift experiments. Mutants that are thermolabile only up until the beginning of DNA synthesis are considered to possess an initiation block, and mutants that are thermolabile throughout the DNA synthetic period are considered to harbor replication blocks. It is easy to imagine situations in which a replication mutant would be misclassified as an initiation mutant by this criterion. For example, if the mutant was temperature sensitive for the synthesis of a protein that functioned in replication, and if this enzyme was synthesized at the beginning of the DNA synthetic interval, then the mutant would appear as an initiation mutant by the criteria used above. Secondly, if the mutant had a leaky defect or a thermolabile protein that slowly inactivated, it would be possible for a defective replication enzyme to appear like an initiation defect. The only argument that can be raised against these possibilities is the fact that several independently isolated alleles have been found for two of the genes classified as having roles in initiation and none of these alleles have execution points after bud emergence (L. H. Hartwell et al., manuscript in press). It would not be expected that all independently isolated alleles would have the same degree of leakiness or that all would be temperature sensitive for synthesis, although these possibilities can not be rigorously ruled out at this time. Only one allele of cdc 8 is known. It is interesting to compare the number of genes found for the initiation of DNA synthesis and DNA replication in yeast with the number anticipated on the basis of our current understanding of these processes. Little is known about the process of initiation of DNA synthesis except that the synthesis of protein is required for this process in bacteria (9), and that recent evidence suggests that RNA polymerase may also be involved (). However, initiation could be quite complex since it is the step at which growth control is exerted in eukaryotes. That is, an initiation mutant might be defective in any process required for the start of the new cell cycle. Mutants defective in two of the initiation genes in yeast (cdc and 7) undergo bud emergence despite their block in initiation of DNA synthesis. Since these mutants complete one of the first two events in the cell cycle, their defects appear to be specific for initiation of DNA synthesis. Mutants defective in the third initiation gene (cdc 8) do not bud at the restrictive temperature and this gene might, therefore, be thought of as being involved in the "start" of the cell cycle rather than specifically in the initiation of DNA synthesis. Two genes

8 VOL. 5, 973 DNA SYNTHESIS GENES IN S. CEREVISIAE 973 a HOU RS FIG. 6. DNA synthesis after a shift to the restrictive temperature during the S period in mutant (cdc 8-). An asynchronous culture of cells was adjusted to 5 x cells/ml and incubated in the presence of a factor for h at 3 C. The a factor was removed by filtration; the cells were resuspended in 8 ml of medium at 3 C containing MUCi of 'C-uracil per ml (5 mci/mmole). At 3 min the culture was divided into 3 portions: one was left at 3 C (); the second was incubated at 3 C in the presence of,g of cycloheximide per ml (A); and the third was incubated at 38 C (a). Samples were removed at various times and analyzed for incorporation of radioactivity into DNA. (dna A and C) have been identified in Escherichia coli as being necessary for the initiation of DNA synthesis (). Five genes (dna B, D, E, F, and G) are thought to be required for DNA replication (elongation) in E. coli (). The dna E gene codes for DNA polymerase III (, ), and dna F codes for the Bi subunit of ribonucleotide reductase (3). One would, therefore, certainly expect more than two replication genes in yeast. There are obvious explanations for our failure to detect more replication genes. Even though the 3 cell cycle genes that have been identified have an average of.6 mutations per gene, the distribution of mutations within genes is nonrandom, and it may be that many cell cycle genes remain to be detected because they are HOU RS FIG. 7. DNA synthesis after a shift to the restrictive temperature during the S period in mutant 8 (cdc 7-). The protocol was the same as in Fig. 6, except that the restrictive temperature was 36 C. 3 H O U R S FIG. 8. DNA synthesis after a shift to the restrictive temperature during the S period in mutant 6--3 (cdc -). The protocol was the same as in Fig. 6, except that the culture was divided into three portions at 6 min, and the restrictive temperature was 36 C.

9 97 HARTWELL J. B ACTERIOL. unlikely to mutate to temperature sensitivity. Furthermore, if redundant copies of any particular gene were present in the genome, it would not be possible to get recessive mutations in these genes. Finally, it should be noted that we have examined DNA synthesis in mutants from only of the 3 genes. The reason for the restriction of our attention to this subset of genes is that all the mutations in the remaining, genes fail to stop division within one cycle after the shift to the restrictive temperature. It may be that within the remaining genes there are mutations that for one reason or another result in leaky blocks in DNA replication. ACKNOWLEDGMENTS The technical assistance of Marilyn Culotti and the critical suggestions of Walton L. Fangman are gratefully acknowledged. This work was supported by a grant from the National Institutes of General Medical Sciences. ADDENDUM IN PROOF Other evidence supporting the notion that protein synthesis is required for the initiation of s. cerevistae DNA synthesis but not for its replication was recently presented (. H. Williamson, Heredity 3: 58, 973). LITERATURE CITED. Biicking-Throm, E., W. Duntze, L. H. Hartwell, and T. R. Manney Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell. Res. 76:99-.. Culotti, J., and L. H. Hartwell. 97. Genetic control of the cell division cycle in yeast. III. Seven genes controlling nuclear division. Exp. Cell. Res. 67: Fuchs, J. A., H.. Karlstriim, H. R. Warner, and P. Reichard. 97. Defective gene product in dna F mutant of Escherichia coli. Nature N. Biol. 38: Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler, and C. Barnoux. 97. Analysis of DNA polymerases II and III in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc. Nat. Acad. Sci. U.S.A. 68: Grossman, L. I., E. S. Goldring, and J. Marmur Preferential synthesis of yeast mitochondrial DNA in the absence of protein synthesis. J. Mol. Biol. 6: Hartwell, L. H. 97. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. : Hartwell. L. H. 97. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J. Mol. Biol. 59: Hartwell, L. H., J. Culotti, and B. Reid. 97. Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proc. Nat. Acad. Sci. U.S.A. 66: Lark, K. G Initiation and control of DNA synthesis. Annu. Rev. Biochem. 38: Lark, K. G. 97. Evidence for direct involvement of RNA in the initiation of DNA replication in Escherichia coli 5T-. J. Mol. Biol. 6:7-6.. Niisslein, V., B. Otto, F. Bonhoeffer. and H. Schaller. 97. Function of DNA polymerase III in DNA replication. Nature N. Biol. 3: Wechsler, J. A., and J. D. Gross. 97. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 3: 73-8.