Vol. 10, No. 11

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 5707-5720 0270-7306/90/115707-14$02.00/0 Copyright C) 1990, American Society for Microbiology

The Phenotype of the Minichromosome Maintenance Mutant mcm3 Is Characteristic of Mutants Defective in DNA Replication SUSAN

I.

GIBSON, RICHARD T. SUROSKY,t AND BIK-KWOON TYE* Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 Received 8 May 1990/Accepted 31 July 1990

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation on the left arm of chromosome V approximately 3 kb centromere proximal of anpl. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 proteins may play interacting roles in DNA replication.

Gel retardation and filter binding assays have been used to identify factors that bind to ARSs. ABF1, identified by its affinity to ARSI, binds to a subset of ARSs and to some sequences that are not associated with ARSs (8, 15). OBF1 (19, 20), identified by its affinity to ARS121, has a DNAbinding specificity similar to that of ABF1, suggesting that these two ARS-binding proteins may be the same (S. Eisenberg, personal communications). Analysis of the OBF1binding site in ARS121 indicates that this site acts as an enhancer of DNA replication in such a way that stimulation of origin function can be provided by a synthetic OBF1binding site independent of its orientation or distance 5' from the ARS consensus sequence (71). The role of ABF1 or OBF1 in DNA replication is unclear, since deletion of its binding sites in a number of ARSs causes only a small decrease in plasmid stability (35, 59, 72). Little is known about the third ARS-binding protein, ABF2, which binds to several sites in ARSI and has an as yet undefined DNAbinding specificity (15). We have taken a genetic approach to identify factors that interact with ARSs (40). This method involves screening for mutants that are unable to stably maintain minichromosomes that contain an ARS and a centromere. Minichromosome maintenance-defective (mcm) mutants representing 18 different complementation groups were isolated during three independent screening procedures (this work; 22, 40). We have focused our study on the characterization of three mcm mutants, mcml-l, mcm2-1, and mcm3-1, from three complementation groups. These mutants affect minichromosome maintenance in an ARS-specific manner. Although these three complementation groups were isolated by three independent mutant screens, each involving a minichromosome carrying a different ARS, the three mutants have similar ARS specificities. In other words, ARSs that confer stability to a minichromosome in one of these mutant strains also confer minichromosome stability in the other strains. Similarly, ARSs that fail to confer minichromosome stability in one of these strains fail to do so in all of these strains. The ARS specificities of these mutants suggested that these

Eucaryotes contain multiple chromosomes, with replication initiating at numerous sites along each chromosome (24). For the yeast Saccharomyces cerevisiae, the total number of replication origins has been estimated by electron microscopy and fiber autoradiography to be between 160 and 400 per haploid genome (45, 50, 51). Autonomously replicating sequences (ARSs) that allow the extrachromosomal maintenance of sequences that contain them have been isolated (28, 63). Studies using electron microscopy (55) and two-dimensional gel systems (5, 30, 31) indicate that ARSs serve as in vivo origins of replication on plasmids and on chromosomes. The nucleotide sequences of more than 20 ARSs have been determined by several groups (3, 34, 68). Comparison of the nucleotide sequences of these ARSs indicates that they are all A+T rich and contain an 11-bp consensus sequence (6). Deletion analysis of some of these ARSs shows that the consensus sequence is essential but not sufficient for ARS activity (3, 9, 34). Flanking sequences 3' and 5' to the consensus sequence also play auxiliary roles in the initiation of DNA replication at ARSs. However, these flanking sequences are very different in the ARSs analyzed, and they lack significant secondary structures such as direct or inverted repeats. Experiments to reconstruct functional ARSs on plasmids suggest that flanking sequences that facilitate the melting or bending of ARSs are important for the initiation of DNA replication at ARSs (69, 73). Local melting of DNA at replication origins by initiator and auxiliary proteins has been demonstrated in the studies of replication origins in Escherichia coli (1), bacteriophage lambda (17), and simian virus 40 (13). It seems obvious that the key to understanding replication initiation at ARSs is to identify initiator and auxiliary proteins that bind to ARSs. * Corresponding author. t Present address: Plant Research Laboratory, Michigan State University, East Lansing, MI 48824. t Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.

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MOL. CELL. BIOL. TABLE 1. Yeast strains

Strain

8534-8C 8534-10A A2 A3 F808 Y133 Y134 K396-27B F483 6697-1 B5816 SUH-H2 DBY4962 R61b R61-lDc R61-2Bd tBR61-2B R61-3Cd tAR61-3C tBR61-3C RY2Be RY2Ce RY6Ae RY71Ae RY8Ae RY2B/Y4 RY6A/Y4 RY6A/8A RY2B/2C RY2B/8A RS2Af

RSlOlBf RS2A/R2 RS2A/tBR2 RS1O1B/R3 RS1O1B/tAR3 8C/1OA 8C/SU 1OA/RY71A

tA1OA/RY71A

Description or genotype'

reference

a leu2-3,112 ura3-52 his4-A34 a leu2-3,112 ura3-52 his4-A34 a leu2-3,112 his3-11,15 a leu2-3,112 his3-11,1S a leu2-3,112 ura3-52 his4-519 adel-100 GAL] a ura3-52 his3-A200 Iys2-801 ade2-101 Atrpl a ura3-52 lys2-801 ade2-101 Atrpl tyri a leul ura3-1 his] adel lys7 met3 AtrpS a ura3 his4 Iys2 thrl adel cdc6 a met4 a ura3-1 leul-12 trp2-1 lys2-1 cycl CYC7H3 a met3 MAL2 trpl URA3::his4 a ade2 his3-A200 ura3-52 leu2-AJOJ::URA3::leu2-A102 lys2-AJO::HIS3::Iys2-A102 a leu2-3,112 ura3-52 his4-A34 mcm3-1 a leu2-3,112 ura3-52 his4-A34 mcm3-1 a leu2-3,112 ura3-52 his3-11,15 mcm3-1 R61-2B with 1 copy of M3LL (-+4100) (LEU2 MCM3) integrated at the mcm3 locus a leu2-3,112 ura3-52 his4-A34 mcm3-1 R61-3C with several copies of M3LL (-+4100) (LEU2 MCM3) integrated at the mcm3 locus R61-3C with YIp5/MCM3 (URA3 MCM3) integrated at the mcm3 locus a ura3-52 his3-A200 ade2-101 mcm3-1 a leu2-3,112 ura3-52 his3-A200 his4-A34 lys2-801 ade2-101 Atrpl mcm3-1 a ura3-52 his4-A34 ade2-101 Atrpl a ura3-52 leu2-3,112 his3-A200 lys2-801 ade2-101 Atrpl a ura3-52 his3-A200 lys2-801 ade2-101 mcm3-1 Diploid formed by crossing RY2B with Y134 Diploid formed by crossing RY6A with Y134 Diploid formed by crossing RY6A with RY8A Diploid formed by crossing RY2B with RY2C

G. Fink G. Fink G. Fink G. Fink G. Fink R. Davis R. Davis S. Klapholtz

Diploid formed by crossing RY2B with RY8A a ura3-52 met3 trpl his4:: URA3 mcm3-1 a ura3-52 his4:: URA3 mcm3-1 Diploid formed by crossing RS2A with R61-2B

Diploid formed by crossing RS2A with tBR61-2B Diploid formed by crossing RS1O1B with R61-3C Diploid formed by crossing RS1O1B with tAR61-3C Diploid formed by crossing 8534-8C with 8534-1OA Diploid formed by crossing 8534-8C with SUH-H2 Diploid formed by crossing 8534-1OA with RY71A 1OA/RY71A with 1 copy of MCM3 gene replaced by interrupted MCM3 gene from M3L (-+500) = LEU2::mcm3

42 64 C. Chan This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

aFor descriptions of plasmids within yeast strains, see Table 2. b Original mcm3-1 mutant isolated after ethyl methanesulfonate mutagenesis of 8534-8C. c Segregant from the cross R61 x A3. d Segregant from the cross R61-1D x A2. Segregant from the cross R61-3C x Y133. f Segregant from the cross R61-3C x SUH-H2.

MCM gene products play a role in minichromosome replication rather than segregation or other functions required for stability. Analysis of the properties and functions of the MCMJ gene product indicates that it is a multifunctional protein. Aside from an involvement in ARS function, Mcml also acts as a transcription factor (48, 49). In this respect, it is similar to the transcriptional silencer proteins ABF1 and RAP1 (8, 59), which show striking sequence homology to each other (16). They are both involved in the repression of the yeast silent mating-type loci and have been shown to bind to the ARSs associated with these loci. Less is known about the Mcm2 and Mcm3 proteins. This report describes the characterization of the mcm3-1 mutant and analysis of the MCM3 gene. Like mcm2-1 (61), this mutant shares many of the properties characteristic of mutants that are defective in some aspect of DNA replication.

MATERIALS AND METHODS Strains and plasmids. E. coli HB101 (hsdS20 recA13) was used for routine bacterial transformations and plasmid preparations. E. coli JM101 [A(lac-proAB) F' traD36 proAB lacIq ZAM15] was used for phage transformations and the preparation of single-stranded DNA (ssDNA). E. coli RR1 (hsdS20) was used for producing the TrpE-Mcm3 fusion protein. E. coli RDP146 [recA Spr A(lac-pro)] and NS2114 Sm (recA Smr, lambda cre prophage in chromosome) were used for shuttle mutagenesis (57). The S. cerevisiae strains used are listed in Table 1. Table 2 describes the plasmids used. Media, chemicals, and enzymes. T4 DNA polymerase, terminal deoxyribonucleotidyltransferase, and the subcloning primer RD22 (International Biotechnologies, Inc., New

PROTEIN INVOLVED IN ARS FUNCTION

VOL. 10, 1990

TABLE 2. Plasmids Plasmid

YCpl YCp131 YCp121 YCp131C YCplOl YCp2B pYES3 YCp5O YCp5O bank YIp5 R61-1 R61-4 YIp5/MCM3 pAB83 pATH3 p3MCM3HH

p3MCM3BC

pCGS286 B620 B620/MCM3 pLB101

pOX38::m-Tn3 pHSS6 pHSS6/MCM3 M3LL(- +4100)

M3L(-+500)

Relevant sequences

or Reference source

ARSI CEN5 LEU2 URA3 ARSJ31 CEN5 LEU2

40 40 40 ARS121 CEN5 LEU2 ARS131C CEN5 LEU2 URA3 40 ARSJ CEN5 LEU2 This laboratory ARS2B CEN5 LEU2 URA3 This study 41 ARS1 CEN5 URA3 54 ARSI CEN4 URA3 54 Yeast DNA bank in YCp5O URA3 63 This study ARSI CEN4 URA3 MCM3 This study ARSI CEN4 URA3 MCM3 This study URA3 MCM3 42 URA3 ANPI Amino terminus of E. coli A. Tzagoloff trpE Expresses TrpE-Mcm3 fusion This study protein in E. coli Expresses TrpE-Mcm3 fusion This study protein in E. coli GAL] UAS fused to lacZ, 23

2p.m ARS URA3 GAL] UAS 2,um ARS URA3 GAL] UAS fused to MCM3, 21Lm ARS URA3 Cmr, Tn3 transposase Apr, mini-Tn3 Kmr ori MCM3 pHSS6/MCM3 with minitransposon m-Tn3(LEU2 lacZ) inserted 3' of MCM3 ORF pHSS6/MCM3 with minitransposon m-Tn3(LEU2) inserted within the MCM3 ORF

23 This study 57 57 57 This study This study

This study

Haven, Conn.)

were used to make deletions in DNA cloned in M13-based vectors. Genetic techniques and DNA transformation. Meiotic tetrad analysis has been described elsewhere (58). Transformation of yeast cells was done by the spheroplasting method

(27). Mitotic plasmid stability assay. Stability assays measure the cumulative loss of a minichromosome from a yeast culture over about eight generations of growth on nonselective media. Single colonies, containing i107 cells per ml, were picked off plates containing selective medium and used to inoculate 5-ml samples of nonselective medium (YEPD). The cultures were grown to saturation (approximately eight generations of growth) and diluted with water, and samples were plated onto YEPD plates. These plates were then replica plated onto selective medium. Minichromosome stability is expressed as the percentage of cells on the YEPD plates that grew on selective plates (40). The minichromosome loss rate is determined by 1 (FI)l/N, where I is the initial percentage of plasmid-containing cells and F is the percentage of plasmid-containing cells after N generations. DAPI staining. Cell staining with the DNA specific dye 4,6-diamidino-2-phenylindole (DAPI) was carried out ac-

cording to published procedures (74). Flow cytometry. Cells stained with propidium iodide according to the method of Hutter and Eipel (32) were ana-

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lyzed by using an Ortho Diagnostics System 2151 machine with 488-nm excitation. Fluctuation analysis (chromosome loss and recombination assay). Each diploid strain used contains a single copy of the URA3 gene, integrated by gene replacement at the his4 locus on the left arm of chromosome III. A single colony, isolated from a plate containing complete medium lacking uracil, was suspended in water and then plated to single colonies on YEPD plates. Plates were incubated at 14, 23, or 30°C for approximately 25 generations of cell growth (until an average colony contained approximately 5 x 107 cells). Colonies were picked, suspended in water, and spread onto 5-fluoroorotic acid (FOA) (2) and YEPD plates. The total number of cells in a colony was measured by plating efficiency and, in some cases, by direct counting of the cells in the suspension. The number of Ura- cells was determined from the number of colonies that grew on an FOA plate. The observed recombination frequencies were multiplied by 2 since only half, on average, of the recombination events led to loss of the URA3 marker from one of the daughter cells. The rates of chromosome loss and mitotic recombination per cell division were determined by the method of the median as described previously (39). Final values for the rates of chromosomal loss or recombination were the results of 18 to 20 independent assays. Isolation of the mcm3 mutant. The mcm3 mutant was isolated by using a slightly modified version of the mutant screen used by Maine et al. (40). The minichromosome YCp2B (47) was used instead of the minichromosome YCpl or YCp131C, and the mutant screen was conducted at 26 instead of 30°C to allow for the isolation of mutants that are temperature sensitive for growth. DNA and RNA hybridizations of radioactively labeled DNA probes. DNA was transferred from agarose gels to nitrocellulose filters as described by Southern (62) or to Biotrans (Biodyne) filters as suggested by Pall Ultrafine Filtration Corp., East Hills, N.Y. RNA was denatured with glyoxal, run in agarose gels, and transferred to Biotrans filters (65). Hybridizations of filters carrying RNA were performed as described by the manufacturer. 32P-labeled double-stranded DNA probes were prepared by nick translation (53). 32p_ labeled ssDNA probes were prepared by using the hybridization probe primer protocol (29). Sequencing and construction of clones for sequencing. Both strands of the DNA fragment that contains the MCM3 gene plus several hundred base pairs of flanking DNA were sequenced at least once. Overlapping DNA fragments from the MCM3 gene were subcloned into the M13 vectors mpl8 and mp19 (46), and then T4 DNA polymerase was used to construct a series of deletions in each of these MCM3 DNA fragments (11). These constructs were then sequenced by the dideoxynucleotide chain termination method (56). Isolation of TrpE-Mcm3 hybrid protein and preparation of anti-Mcm3 antibodies. E. coli RR1 cells were transformed with the trpE-MCM3 fusion gene p3MCM3HH or p3MCM3BC. Induction and partial purification of TrpE hybrid proteins have been described elsewhere (14, 36). To generate antibodies, a TrpE-Mcm3 hybrid protein that contains 37 kDa from the amino terminus of the TrpE protein fused to -60 kDa of the Mcm3 protein was purified from E. coli extracts and used to immunize rabbits. Three giant Flemish/chinchilla rabbits were immunized with 0.4 to 1.1 mg of TrpE-Mcm3 hybrid protein and Freund complete adjuvant (70). The rabbits were boosted twice, 1 and 6 months after the initial injection, with Freund incomplete adjuvant and 0.3 to 1.0 mg of TrpE-Mcm3 hybrid protein.

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TABLE 3. Stabilities of minichromosomes carrying different ARSs in mutant and wild-type strains at various temperatures Stability of given YCp minichromosomeb Strainsa Genotype Temp (°C)

R61-2B, R61-3C 8534-8C, A2

mcm3 MCM3

RY2B/2C, RY2B/8A

mcm3/mcm3

RY3BIY4, RY6A/8A RY6A/Y4, 8C/10A

mcm3lMCM3 MCM31MCM3

14 23 30 14 23 30 23 23 23

1

131

121

131C

0.3 0.6 0.1 90 89 80 0.2 52 88

0.3 0.2

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking h...
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