Download Genetic Analyses of Schizosaccharomyces pombe dna2

Copyright  2000 by the Genetics Society of America
Genetic Analyses of Schizosaccharomyces pombe dna2ⴙ Reveal That Dna2 Plays an
Essential Role in Okazaki Fragment Metabolism
Ho-Young Kang,*,† Eunjoo Choi,* Sung-Ho Bae,* Kyoung-Hwa Lee,* Byung-Soo Gim,*
Hee-Dai Kim,* Chankyu Park,† Stuart A. MacNeill‡ and Yeon-Soo Seo*
*National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School
of Medicine, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea, †Department of Biological Sciences, Korea Advanced Institute of Science
and Technology, Yusung-Ku, Taejon, 305-701, Korea and ‡Institute of Cell and Molecular Biology, University of Edinburgh,
Edinburgh EH9 3JR, United Kingdom
Manuscript received November 17, 1999
Accepted for publication March 31, 2000
ABSTRACT
In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of
dna2⫹ of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define
its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the
restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis.
Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a
role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2⫹ was disrupted duplicated
their DNA content during germination and also arrested at late S-phase. Inactivation of dna2⫹ led to
chromosome fragmentation strikingly similar to that seen when cdc17⫹, the DNA ligase I gene, is inactivated.
The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes
encoding subunits of polymerase ␦ (cdc1⫹ and cdc27⫹), DNA ligase I (cdc17⫹), and Fen-1 (rad2⫹). Each
of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they
all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of
these results, we conclude that dna2⫹ plays a direct role in the Okazaki fragment elongation and maturation.
We propose that dna2⫹ acts as a central protein to form a complex with other proteins required to
coordinate the multienzyme process for Okazaki fragment elongation and maturation.
A
T the initiation of chromosomal DNA replication,
strand separation occurs to establish replication
forks. Due to the antiparallel structure of double helix
DNA and the conserved 5⬘ to 3⬘ polarity of all DNA
polymerases known to date, one strand (designated the
leading strand) is continuously synthesized in the direction of fork movement. The other strand (the lagging
strand) grows discontinuously in a direction opposite
to fork movement (Kornberg and Baker 1992). The
generation of a continuous DNA strand from the short
and discontinuous lagging-strand fragment can be regarded as the most frequent and yet complex enzymatic
event at replication forks.
Okazaki fragment synthesis requires the action of polymerase (pol) ␣-primase, DNA pol ␦, and/or ε with proliferating nuclear antigen (PCNA) and replication factor-C (RFC; Stillman 1994; Bambara et al. 1997; Baker
and Bell 1998). Pol ␣, tightly complexed with DNA
primase, plays a role in the initiation of DNA synthesis
by providing RNA-DNA primers for both leading and
Corresponding author: Yeon-Soo Seo, National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research
Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku Suwon, Kyunggi-Do, 440-746, Korea.
E-mail: [email protected]
Genetics 155: 1055–1067 ( July 2000)
lagging strands. Pol ␦ is involved in the elongation of
the RNA-DNA primers on the lagging strand template
(Okazaki fragment elongation) as well as the replication
of the leading strand. Pol ␦ (and pol ε) requires two
accessory factors, PCNA and RFC, for its processive DNA
synthesis. Saccharomyces cerevisiae pol ␦ complex is composed of three subunits having apparent molecular
masses of 125, 58, and 55 kD encoded by the POL3,
POL31, and POL32 genes, respectively (Gerik et al.
1998). Studies of Schizosaccharomyces pombe have identified four subunits of pol ␦ that migrate with apparent
molecular masses of 125, 55, 54, and 22 kD that are
encoded by pol3⫹/cdc6⫹, cdc1⫹, cdc27⫹, and cdm1⫹, respectively (MacNeill et al. 1996; Zuo et al. 1997).
Okazaki fragments are ligated together through a process called Okazaki fragment maturation, which requires the combined action of Fen-1 (also called 5⬘ to
3⬘ exonuclease, MF1, or DNase IV), RNase HI, and DNA
ligase I (Ishimi et al. 1988; Goulian et al. 1990; Waga
and Stillman 1994; Waga et al. 1994). In the current
model, RNA primers are removed by Fen-1 (assisted by
RNase HI), followed by gap filling by DNA pol ␦ (and/
or pol ε) and the joining of the nicks by DNA ligase I.
Recently, it was shown that Fen-1 is a structure-specific
endonuclease that cleaves at the junction of a flap structure (Bambara et al. 1997; Lieber 1997). This suggests
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H.-Y. Kang et al.
that branch structures may be generated during Okazaki
fragment metabolism. The mechanism, however, by
which the unannealed branch structure is generated is
yet to be discovered. Moreover, the RAD27 gene (also
called RTH1) encoding S. cerevisiae Fen-1 (yFen-1 or
Rad27) is not essential in vivo, although cells lacking
RAD27 are inviable at certain growth conditions (e.g.,
37⬚; Reagan et al. 1995; Sommers et al. 1995). The RNase
HI gene (RNH35) in S. cerevisiae is not required for
either DNA replication or cell growth (Frank et al.
1998). Instead, the deletion of RAD27 increased the
rates of spontaneous mutation, mitotic recombination,
and chromosome loss (Johnson et al. 1995; Reagan et
al. 1995; Vallen and Cross 1995), consistent with it
having critical roles for chromosome maintenance
(DeMott et al. 1996, 1998; Klungland and Lindahl
1997; Tishkoff et al. 1997; Freudenreich et al. 1998;
Kim et al. 1998; Gary et al. 1999a; Wu et al. 1999). These
results deemphasize the only known role of Fen-1
for DNA replication and strongly argue for the existence
of an alternative enzymatic system that allows cells to
endure the loss of Fen-1/RNase HI functions.
Genetic studies in S. cerevisiae uncovered a component
likely to be involved in Okazaki fragment maturation
by virtue of its genetic and physical association with
Fen-1 (Budd and Campbell 1997), adding further complexity to Okazaki fragment processing. The essential
DNA2 gene of S. cerevisiae encodes a 172-kD protein with
characteristic DNA helicase motifs (Budd and Campbell 1995; Budd et al. 1995). DNA2 homologs are found
throughout eukaryotes including humans, plants, fish,
and nematodes (Budd and Campbell 1997; Formosa
and Nittis 1999), suggesting that its role may be evolutionarily conserved in all eukaryotes. A specific association of yFen-1 and S. cerevisiae Dna2 was demonstrated
both genetically and biochemically (Budd and Campbell 1997). Cells harboring temperature-sensitive (ts)
alleles of S. cerevisiae DNA2 arrested at either G2/M
with a 2C DNA content at the restrictive temperature
(Fiorentino and Crabtree 1997) or at S phase (Budd
and Campbell 1995), depending on the Dna2 alleles
used. It was also speculated that Dna2 displaces RNA
primers from template DNA by translocating along the
template DNA as does pol ␦, creating a flap-like substrate
for Fen-1 endonuclease (Bambara et al. 1997; Waga
and Stillman 1998). This provided a possible mechanism by which Dna2 is involved in Okazaki fragment
maturation. Recently, however, we showed that the recombinant S. cerevisiae Dna2 protein intrinsically contained a strong single-stranded DNA-specific endonuclease activity (Bae et al. 1998), providing a possible
function for Dna2 in Okazaki fragment maturation. Unfortunately, this process is poorly understood and remains to be defined more clearly.
To test this possibility, we characterized the endonuclease activity of S. cerevisiae Dna2 and found that Dna2
possessed many enzymatic activities capable of removing
the 5⬘ primer oligoribonucleotides (S.-H. Bae and Y.-S.
Seo, unpublished results). In addition to a biochemical
approach, we sought in vivo evidence for a role of DNA2
in Okazaki fragment metabolism. For this purpose, we
isolated the S. pombe homolog (dna2⫹) of S. cerevisiae
DNA2 and constructed ts alleles of dna2⫹. Characterization of the S. pombe dna2 mutants revealed that S. pombe
Dna2 interacted genetically with Cdc1 and Cdc2 (subunits of pol ␦), Rad2 (S. pombe homolog of yFen-1), and
Cdc17 (DNA ligase I). All of these gene products are
essential for either elongation or maturation of Okazaki
fragments. Our results extend the previous observations
to another organism and present new in vivo data that
dna2⫹ is directly involved in Okazaki fragment metabolism. On the basis of our genetic studies, we propose
a novel mechanism by which Dna2 participates as a
component of a multienzyme complex for the synthesis
and processing of Okazaki fragments.
MATERIALS AND METHODS
Strains and growth media: The following S. pombe strains
were used in this study (Table 1). The haploid strain HK100
(h⫺ ura4-D18 leu1-32) was used to isolate the temperaturesensitive mutants. The diploid strain EC1 (h⫹/h⫺ leu1-32/leu132 ura4-D18/ura4-D18 ade6-M210/ade6-M216) was constructed by mating ED666 (h⫹ leu1-32 uraD-18 ade6-M210) and
ED667 (h⫺ leu1-32 uraD-18 ade6-M216) and was used for gene
disruption (ED666 and ED667, gifts from Dr. J. Rho, Seoul
National University, Korea). The h⫺ haploid strains with either
cdc17-k42 or cdc24-M38 (Nasmyth and Nurse 1981) and
rad2::ura4⫹ alleles (gifts of Dr. J. Murray, University of Sussex,
UK) were used to evaluate the effects of combining mutations
(synthetic lethality) with dna2-C2. The haploid strains carrying
hus1-14 (Enoch et al. 1992) or rhp9::ura4⫹ (Willson et al.
1997; gifts from Dr. F. Z. Watts, University of Sussex, United
Kingdom) were used to construct the dna2-C2 hus1-14 or dna2C2 rhp9::ura4⫹ strain, respectively. S. pombe cells were grown
either in YE or Edinburgh minimal medium (EMM) media
supplemented with appropriate nutrients (Alfa et al. 1993).
Transformation of S. pombe was performed as described (Prentice 1992). For constructions of strains used in this study
(Table 1), standard S. pombe genetic methods were used (Moreno et al. 1991).
DNA, oligonucleotides, plasmids, and libraries: All PCR
primers or oligonucleotides used were commercially synthesized (BioServe Biotechnologies, Laurel, MD). Primers A and
B (degenerate primers; 5⬘-GGN-ATG-CCN-GGN-ACN-GGNAAR-ACN-AC-3⬘ and 5⬘-DAT-RTT-GTC-NAC-NGC-RCT-RTGNGT-RTA-3⬘, respectively) were used to amplify the dna2⫹
gene fragment of S. pombe. Primers C and D (5⬘-CGG GAT
CCA TAT GGA TTT TCC AGG TCT G-3⬘ and 5⬘-CCG CTC
GAG AAT TAA GCA AAC TAA GCT-3⬘, respectively) were
used to amplify cdc24⫹ and primers E and F (5⬘-CGG GAT
CCT TAT GCG AAC AGT ATT TTC G-3⬘ and 5⬘-CCG CTC
GAG TCA GCA GTA ACT CTC AGC TA-3⬘, respectively) were
used for cdc17⫹. The primers G and H (5⬘-GAA TTC ATG
GAG GAA TGG AGA AAC TT-3⬘ and 5⬘-CTC GAG TTA TTT
CTT TCC AAA AAA GG-3⬘, respectively) were used to obtain
cdc27⫹. The 54-mer oligonucleotide (5⬘-AAG TAA GAA GTA
TTT TCT TCT TTT TGG CAA GCA ATG ATC TGA TTA
AGC TAG AAA-3⬘) contained the unique internal sequence
of the amplified dna2⫹ fragment and was used as a probe to
screen full-length cDNA or genomic DNA of dna2⫹.
Role of Dna2 in Okazaki Fragment Metabolism
The genomic dna2⫹ gene was cloned into pBluescript
SK(⫹) plasmid (Stratagene, La Jolla, CA) between the EcoRI
and XhoI sites to make pSK-dna2⫹. A 3.9-kb EcoRI-KpnI fragment (Figure 1B, EcoRI in multiple cloning sites of vector and
unique KpnI within dna2⫹) and a 3.1-kb SalI-XhoI fragment
(Figure 1B, unique SalI within dna2⫹ and XhoI in multiple
cloning sites of the vector) from pSK-dna2⫹ were independently cloned into pTZ19R (Pharmacia, Piscataway, NJ) to
construct pSpdna2N and pSpdna2C, respectively. A BamHI
fragment (1.8 kb) from pTZ19R-cdc1EBg⌬U (MacNeill et
al. 1996) and a HindIII fragment (1.8 kb) from pGRP-130 (a
plasmid harboring ura4⫹ gene flanked by HindIII sites) were
cloned into pSpdna2N and pSpdna2C, respectively, to introduce the ura4⫹ selection marker gene into the vectors. These
two plasmids were designated pTZ-dna2N and pTZ-dna2C,
respectively (Figure 1B) and used for random mutagenesis in
vivo. A pUR19-based genomic library (Barbet et al. 1992; a
gift from Dr. H. Ohkura, University of Edinburgh, United
Kingdom) was used to screen for multicopy suppressors of
dna2-C2 mutants.
To construct the plasmid pREP1-dna2⫹ in which dna2⫹ is
placed under the control of the nmt1 promoter, full-length
dna2⫹ cDNA was cloned into the pBlueBacHis2 vector (Invitrogen, Carlsbad, CA) between the BamHI and KpnI sites to
create pBBH-dna2⫹. In this vector, dna2⫹ cDNA is flanked by
two EcoRI sites or the XhoI sites of the vector origin. The XhoI
fragment of dna2⫹ was blunted by the use of Klenow and then
ligated into blunt-ended SalI sites of pREP1.
Cloning of cDNA and genomic DNA and characterization
of its transcript: Degenerate primers were designed from the
conserved regions between DNA2 of S. cerevisiae and its human
homologue open reading frame (Eki et al. 1996). Degenerate
primers A and B (200 pmol each) were used for amplification
of the S. pombe genomic DNA template (600 ng) in 20 ␮l of
reaction buffer (Promega, Madison, WI). Four bands 350,
150, 110, and 75 bp in size were amplified after 12 cycles
(annealing, 1 min at 45⬚) plus 26 cycles (annealing, 1 min at
50⬚) of PCR (extension, 1 min at 72⬚; denaturation, 1 min
at 95⬚) in a thermocycler (MJ Research, Watertown, MA).
Sequencing analysis revealed that the 110-bp PCR-derived
band contained the sequence that is highly conserved with
S. cerevisiae DNA2 (Budd and Campbell 1995). The 54-mer
oligonucleotide from the internal sequence of the 110-bp PCR
product was synthesized and used as a specific probe to screen
a S. pombe cDNA library in pGAD-GH (Clontech, Palo Alto,
CA). Among 1 ⫻ 105 colonies screened, one cDNA clone of
a 970-bp insert was obtained. The insert was radiolabeled and
used as a probe to further screen S. pombe cDNA and genomic
DNA libraries (gifts of Dr. H. Yoo, Korea Research Institute
of Bioscience and Biotechnology) to isolate additional clones
containing the missing 5⬘ and 3⬘ terminus of dna2⫹. Fulllength cDNA and genomic DNA of dna2⫹ were cloned by
repeating these procedures and their sequences were determined with an automated DNA sequencer (ABI PRISM 310
Genetic Analyzer from Perkin-Elmer, Norwalk, CT).
Gene disruption and screening for temperature-sensitive
mutant alleles: The PstI fragment (Figure 1A, 0.8 kb, an internal region of dna2⫹) of pSK-dna2⫹ was subcloned into pBlueScript SK(⫹) to construct pSK-0.8PstI. The HindIII fragment
(1.8 kb, the intact ura4⫹ gene) was isolated from pREP2 (a
gift of Dr. J. Hurwitz at Sloan-Kettering Institute) and inserted
into the unique HindIII site located within the subcloned PstI
fragment in pSK-0.8PstI. The resulting construct was digested
with PstI and introduced into the EC1 diploid strain by electroporation to obtain a dna2⫹ knockout strain. Stable Ura⫹ transformants were isolated and verified for integration of the
marker gene at the desired locus by PCR and genomic Southern analyses.
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TABLE 1
Strains used in this study
Strain
EC1
EC2
EC3
HK10
HK11
HK12
HK13
HK14
HK15
HK16
HK100
Genotype
h⫹/h⫺ leu1-32/leu1-32 ura4-D18/ura4-D18 ade6M210/ade6-M216
h⫹/h⫺ dna2::ura4⫹/⫹ leu1-32/leu1-32 ura4-D18/
ura4-D18 ade6-M210/ade6-M216
h⫹/h⫺ dna2⫹/dna2⫹ leu1-32/leu1-32 ura4⫹/ura4D18 ade6-M210/ade6-M216
h⫺ dna2-C2 leu1-32 ura4-D18
h⫺ dna2-C1 leu1-32 ura4-D18
h⫺ dna2-C2 hus1-14 leu1-32 ura4-D18
h⫺ dna2-C2 rhp9::ura4⫹ leu1-32 ura4-D18
h⫹ cdc17-K42 leu1-32
h⫹ rad2::ura4⫹ leu1-32 aed6-704 ura4-D18
h⫹ cdc24-M38
h⫺ leu1-32 ura4-D18
Except HK100, all strains were derived or constructed from
the original sources described in materials and methods.
Temperature-sensitive dna2 mutants were screened and isolated using the strategy of Francesconi et al. (1993), except
that the gene was mutagenized by amplification of plasmids
in an Escherichia coli mutator strain (Epicurian Coli XL 1-Red;
Stratagene) deficient in three major DNA repair pathways.
The plasmids, pTZ-dna2N or pTZ-dna2C, were introduced
into and amplified in the E. coli mutator strain. The plasmids
recovered were digested with NdeI (pTZ-dna2N) or with NcoI
(pTZ-dna2C; Figure 1B) and were used to transform the
HK100 strain to Ura⫹ on EMM plates supplemented with
leucine at 25⬚. Ura⫹ transformants had integrated the linearized DNA by homologous recombination at the dna2 locus to
produce a complete dna2 gene and a truncated copy separated
by the ura4⫹ gene. Ura⫹ colonies from the pTZ-dna2N and
the pTZ-dna2C (9 ⫻ 103 and 1 ⫻ 104 transformants, respectively) were replica plated on EMM supplemented with leucine
and Phloxin B (1.75 ␮g/ml) and screened for clones that did
not grow at the elevated temperature (37⬚). To recover the
integrated plasmids, the total DNA was isolated from clones
that did not grow at the restrictive temperature. The DNA
was digested with an appropriate restriction enzyme (e.g., NcoI
for pTZ-dna2C integrants; Figure 1), ligated to recircularize
the plasmids, and then introduced into E. coli DH5␣ strain.
The plasmids were recovered from the resulting ampicillinresistant transformants. The sequences of mutant alleles were
also determined.
Analyses of dna2::ura4ⴙ and dna2-C2 cells: An overnight culture (1 ml) of EC2 diploid strain (Table 1) was inoculated
into EMM (200 ml) supplemented with leucine and glutamate
instead of NH4Cl as the nitrogen source and incubated at 30⬚
for 72 hr with shaking. The cells were harvested and washed
with 200 ml of sterile water and then resuspended in sterile
water (200 ml) containing 0.5 ml of Helix promatia juice (Sepracor, France) to digest ascus walls. After incubation at 30⬚ for
18 hr with shaking, the spores were harvested, washed with
100 ml sterile water three times, and then resuspended in 10
ml of sterile water. This suspension was inoculated into 200
ml of EMM supplemented with adenine and leucine (OD595
of ⵑ0.15) and the cultures were incubated with shaking at
30⬚. Samples (10 ml, 100 ␮l) were taken every 3 hr for flow
cytometry and cell number determination, respectively. Samples for DAPI (4⬘,6-diamidino-2-phenylindole) staining were
also taken at 19 hr after inoculation. As a control, wild-type
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H.-Y. Kang et al.
dna2⫹ spores were identically prepared using diploid strain
EC3 (Table 1), which is heterozygous for ura4⫹ (ura4⫹/ura4D18), in which half of the spores produced were dna2⫹ and
uracil prototrophic spores. The analyses of dna2-C2 mutants
were also performed as described above for dna2⫹-deleted
spores except that the culture was grown in EMM supplemented with leucine and uracil and shifted to 37⬚ for the
indicated times before sampling. Flow cytometry analysis and
DAPI staining were performed as described (MacNeill and
Fantes 1994).
Screening for multicopy suppressors of dna2-C2 mutant:
The HK10 haploid strain (Table 1) was grown at 25⬚ to midlog
phase in EMM supplemented with leucine and uracil. The
genomic library in pUR19 was transformed into dna2-C2 mutants, and the cells were plated on EMM plates containing
leucine and were allowed to grow at 25⬚ for 24 hr and at 34.5⬚
for an additional 4–6 days. The temperature-tolerant Ura⫹
transformants were selected and streaked on EMM plates containing leucine at 25⬚. The plasmids were recovered from the
candidate Ura⫹ transformants and checked for their ability
to suppress the ts phenotype by reintroducing them into dna2C2 mutants. The sequences were then determined and analyzed using the BLAST server (http://www.ncbi.nlm.nih.gov/
cgi-bin/BLAST/nph-newblast).
Yeast two-hybrid assays: The EcoRI fragment from pBBHdna2⫹ as described above was inserted into pGBT9 (Clontech)
for GAL4 DNA-binding domain fusion. The cdc24⫹ cDNA was
amplified from an S. pombe cDNA library (a gift of Dr. H. Yoo,
Korea Research Institute of Bioscience and Biotechnology)
using primers C and D (complementary to the 5⬘- and 3⬘-end
of cdc24⫹ and containing BamHI and XhoI sites, respectively).
The amplified cdc24⫹ cDNA was directly cloned into pCR2.1
TA cloning vector (Invitrogen) to construct pCR2.1-cdc24⫹;
DNA sequencing was carried out to assure that there was no
erroneous nucleotide inserted in cdc24⫹ cDNA during PCR
amplification. The BamHI-XhoI fragment (1.5 kb) of cdc24⫹
cDNA was then inserted into pGAD424 between BamHI and
SalI (compatible with XhoI) sites to make pGAD424-cdc24⫹.
The PCR amplification of cdc17⫹ cDNA (using primers E and
F) and the construction of pGAD424-cdc17⫹ were carried out
using the same strategy as for cdc24⫹. Using primers G and
H, pGAD424-cdc27⫹ containing cdc27⫹ cDNA was also made
using the same strategy for cdc24⫹ except that the 5⬘ primer
(primer G) contained an EcoRI site instead of BamHI. The
BamHI restriction fragment from pET28c-cdc1⫹ (a gift from
Dr. J. Hurwitz at Sloan-Kettering Institute) was cloned into
the BamHI site of pGAD424 to construct pGAD424-cdc1⫹. The
pACT2-rad2⫹ plasmids were obtained from Dr. J. Murray (University of Sussex, United Kingdom). S. cerevisiae Y190 strain was
transformed using the lithium acetate method as described
(Gietz et al. 1992) and Leu⫹ Trp⫹ colonies were selected on
SD plates. The transformants were transferred to Whatman
filter papers (cat. no. 1005 090) presoaked with Z buffer
(Miller 1972) containing ␤-mercaptoethanol (38.6 mm) and
X-gal (0.33 mg/ml). The filter papers were frozen at ⫺70⬚
or in liquid nitrogen and thawed at room temperature to
permeabilize the cells. The filter papers were then placed
on another presoaked filter and incubated at 30⬚ until the
appearance of blue color. The colonies exhibiting positive
blue colors were then picked from original plates and restreaked on SD plates. The ␤-galactosidase assays were performed using liquid cultures as described (Miller 1972; Kaiser et al. 1994).
RESULTS
Isolation and structure of the dna2ⴙ gene: The dna2⫹
gene, an S. pombe homolog of budding yeast DNA2, was
Figure 1.—Structure of the dna2⫹ gene and positions of
dna2 ts mutations. (A) The dna2⫹ exons are indicated by four
open bars, and the three introns are denoted by thin lines
(intron 1, nucleotide positions 359–403; intron 2, 3198–3246;
intron 3, 3962–4008). The numbers 1 and 4334 on the scale
bar above the dna2⫹ gene refer to the adenine of the start
codon (AUG) and the last nucleotide of the stop codon
(UGA), respectively. For the construction of dna2⫹-disrupted
mutants, the PstI fragment (indicated by two wedges above
the second exon) was replaced by the ura4⫹ gene as described
in materials and methods. The positions of dna2-C1 (C to
T; Pro956 to Leu) and dna2-C2 (T to C; Leu1079 to Ser) are
denoted by asterisks above the open reading frame. The five
solid bars within exons and roman numerals below stand for
the conserved helicase motifs from 21 related proteins (Hodgman 1988). The two shaded bars, designated by lowercase
roman numerals (i and ii) in the second exon, indicate conserved amino acid sequences in the N-terminal half among
three Dna2 homologs from humans, budding yeast, and fission
yeast. (B) Two thick lines represent the fragments used to
construct plasmids pTZ-dna2N and pTZ-dna2C for mutagenesis of the dna2⫹ gene to obtain conditionally lethal mutants.
The arrows indicate the enzyme sites used to construct ts
mutants (NdeI, nucleotide position 1728; SalI, 1852; NcoI, 2569;
and KpnI, 2719). (C) The amino acid sequences of conserved
helicase motifs in S. pombe Dna2 are presented using the singleletter code. The identical amino acid sequences conserved
among the three Dna2 ORFs from humans and two yeasts are
shown as boldface capital letters and their positions in S.pombe
Dna2 are indicated in parentheses. The DNA sequence of
dna2⫹ was deposited in GenBank as accession no. AF144384.
cloned by PCR amplification and repeated cycles of
standard screening procedures as described in materials and methods. Both genomic and cDNA sequences
of dna2⫹ have been deposited into GenBank under accession no. AF144384. Alignment of nucleotide sequences from genomic and cDNA clones showed that
the open reading frame was interrupted by three introns
[nucleotide positions starting from adenine (⫹1) of the
initiation codon, 359–403; 3198–3246; and 3962–4008;
Figure 1A). Computer analysis identified a single open
reading frame (ORF) of 4191 nucleotides that encoded
a 158-kD protein with 1397 amino acids. In support of
this, we detected the 4.6-kb mRNA transcript by North-
Role of Dna2 in Okazaki Fragment Metabolism
ern blot analysis (not shown). While this study was in
progress, an identical gene was isolated as a multicopy
suppressor of the cdc24-G1 ts mutant and named dna2⫹
on the basis of its significant homology with S. cerevisiae
DNA2 (Gould et al. 1998). Recently, the complete sequence of the dna2⫹ gene was released from the Sanger
Center (GenBank accession no. CAB38508), but contained an ORF one amino acid longer than the one we
cloned. The extra amino acid reported by the Sanger
Center might result from a computer prediction that
chose the first splicing acceptor site in the first intron
among two possible candidate acceptor sites, adding
one amino acid at position 120. The S. pombe Dna2
protein contains conserved helicase motifs I, II, III, V,
and VI, characteristic of helicases (Hodgman 1988;
Gorbalenya et al. 1989); these motifs are localized to
the C-terminal one-third of the protein (Figure 1, A
and C).
The dna2ⴙ gene product is essential, but not required
for initiation and elongation stages of DNA replication
during germination of spores: We investigated whether
S. pombe dna2⫹ is essential for cell viability by disrupting
dna2⫹ using S. pombe strain EC1 (Table 1), as described
in materials and methods. A dna2::ura4⫹/dna2⫹ heterozygous diploid strain (EC2, Table 1) was sporulated
on malt extract agar (ME) plates. Tetrad analyses of the
resulting asci reproducibly yielded two viable spores,
both of which were Ura⫺ (9 out of 10 tetrads tested; 1
tetrad showed only one viable Ura⫺ spore), indicating
that dna2⫹ is an essential gene like S. cerevisiae DNA2
(Budd and Campbell 1995; not shown). We examined
the phenotype of the dna2⫹-disrupted spores obtained
from the heterozygous EC2 diploid strain. Spores
formed from EC2 were inoculated into EMM lacking
uracil. Under this growth condition, only spores possessing the disrupted dna2::ura4⫹ gene could germinate
and grow. Mutant cells taken at the 19-hr time point
were stained with DAPI and examined microscopically
for their morphology (Figure 2A). Most dna2::ura4⫹
cells at this stage were highly elongated and mononuclear, in keeping with a typical cell division cycle (cdc)
mutant phenotype (Figure 2A).
Since the Dna2 protein in S. cerevisiae was suspected
to have an important role in DNA replication (Budd
and Campbell 1995, 1997; Budd et al. 1995), we examined the changes in DNA content of the dna2⫹-deleted
spores during germination. In this experiment, wildtype dna2⫹/dna2⫹ diploid cells (EC3, Table 1) heterozygous for ura4⫹ (ura4⫹/ura4-D18) were used as a positive
control and processed identically along with EC2 diploid cells. Spores obtained from both diploid strains
were allowed to germinate for 6 hr, after which time
samples were taken every 3 hr up to 18 hr. The cells
were stained with propidium iodide for flow cytometry
analyses to investigate their DNA content. As shown in
Figure 2B (left), DNA replication of wild-type spores
(dna2⫹) was initiated 6–9 hr after inoculation and com-
1059
Figure 2.—The dna2⫹ gene is not required for bulk DNA
synthesis when cells are germinating or growing vegetatively.
(A) Wild-type and dna2⫹-disrupted cells were stained with
DAPI and examined with fluorescence microscopy. (B) Flow
cytometric analysis of wild-type and dna2⫹-disrupted spores
during germination. Wild-type dna2⫹/dna2⫹ diploid cells heterozygous for ura4⫹ (ura4⫹/ura4-D18) as a positive control
were identically processed along with dna2⫹/dna2::ura4⫹ diploid cells. Spores obtained from both diploid strains germinated for 6 hr, after which their DNA content was determined
by flow cytometry as described in materials and methods.
The positions of two DNA peaks (unreplicated and fully replicated) are indicated as 1C and 2C. (C) Wild-type and dna2C2 cells incubated at 37⬚ for 6 and 8 hr were stained with
DAPI and examined under fluorescence microscopy.
pleted by 15 hr. Like wild type, the mutant spores
(dna2::ura4⫹) were capable of initiating DNA replication, but their rate of replication was slower and completed 18 hr after inoculation (Figure 2B, right).
Isolation and characterization of temperature-sensi-
1060
H.-Y. Kang et al.
tive mutants: To examine the effect of mutations of
dna2⫹ in vegetatively growing cells, we constructed conditional mutants of dna2⫹ and investigated their phenotype. Two putative dna2 ts mutants were obtained from
cells (HK100) transformed with a linearized plasmid
pTZ-dna2C that had been in vivo mutagenized (Figure
1, A and B). The verification that these mutants were
ts was carried out as follows (not shown, unless indicated
otherwise): (i) The ura4⫹ marker was stably maintained
and tightly linked to the temperature lethality; (ii) the
plasmid, pREP1-dna2⫹ containing the wild-type cDNA
of dna2⫹, was able to rescue the ts mutants (Figure
5; in addition, the plasmid pTZ-dna2C that was not
subjected to mutagenic treatment was able to abolish
the ts phenotype when integrated into the chromosome
of candidate mutants); and (iii) plasmids recovered
from the putative mutants reestablished the ts phenotype when introduced into wild-type cells after being
linearized. The two ts mutants isolated satisfied all of
these criteria, establishing that they contained mutations associated specifically with the chromosomal
dna2⫹ gene. These two ts mutants were named dna2-C1
and dna2-C2 (Figure 1A). The mutations were C-G to
T-A (dna2-C1) and T-A to C-G (dna2-C2) transitions,
resulting in the alteration of amino acid residue Pro956
to Leu and Leu1079 to Ser, respectively. The two residues
Pro956 and Leu1079 are conserved from budding yeast to
human, and Pro956 is located in the nucleotide-binding
motif (Figure 1A). At the permissive temperature, cells
carrying the dna2-C1 allele showed a slight growth defect, whereas those with the dna2-C2 allele showed no
differences in growth, compared to wild-type cells (not
shown). Following a shift to the nonpermissive temperature (37⬚ for 6–8 hr), dna2-C2 cells arrested as elongated
cells with a single nucleus (Figure 2C) that doubled
their DNA content (measured by FACScan analyses, not
shown). Cells carrying dna2-C1, subjected to the same
analysis, yielded identical results (not shown). These
findings are strikingly similar to those obtained with
dna2::ura4⫹ disrupted spores (Figure 2A) and indicate
that bulk chromosomal DNA replication occurs in the
absence of a functional dna2⫹ product. These results
are in accordance with those obtained for S. cerevisiae
DNA2 (Fiorentino and Crabtree 1997), suggesting
that the in vivo function of Dna2 is conserved in the
two yeasts. We concluded that the dna2⫹ gene of S.
pombe is not required for the initiation and elongation
of replication forks, essential steps to replicate the chromosomal DNA en masse, regardless of modes of growth.
The temperature-sensitive mutation causes impaired
resistance to distinct genotoxic agents: Since inactivation of the dna2⫹ gene product did not affect DNA
synthesis, we investigated the effects of genotoxic agents
on HK10 dna2-C2 mutant strain (Table 1) in an effort
to gain insight into the role(s) played by the dna2⫹
gene in DNA transactions other than replication. The
experiments were done at the semipermissive tempera-
Figure 3.—Cells carrying a dna2-C2 mutant allele are sensitive to alkylating agents and a DNA replication inhibitor. Cells
with either wild-type (wt) or mutant (dna2-C2, designated by
C2) alleles of dna2⫹ were grown on minimal medium containing the indicated concentration of drugs at the permissive
temperature (28⬚). The number of wild-type or mutant cells
was first determined, and then serially diluted samples (104,
103, 102, and 10 cells) were spotted and grown for 4 days
on plates containing the indicated concentrations of drugs,
methyl methanesulfonate (MMS) or hydroxyurea (HU).
ture of 28⬚, which does not affect the growth of the
dna2-C2 mutant (Figure 3). The dna2-C2 mutant was
sensitive to methylmethane sulfonate (MMS) and
slightly sensitive to 10 mm hydroxyurea (HU) but not
to UV (doses ranging from 0 to 400 J/m2) compared
to wild type (Figure 3). In view of its remarkable sensitivity to MMS, an alkylating agent, dna2⫹ is likely to play
an important role in DNA repair, although the mechanism by which this occurs is unclear. The HK11 strain
containing the dna2-C1 allele (Table 1) responded similarly to the various genotoxic agents tested above, like
the dna2-C2 mutant (not shown).
The absence of dna2ⴙ function triggers the replication
checkpoint: To test whether dna2⫹ is involved in DNA
replication, we constructed a strain containing both
dna2-C2 and hus1-14 mutations. The hus1⫹ gene plays a
role in the DNA replication checkpoint: hus1-14 mutant
cells with unreplicated DNA or damaged DNA fail to
arrest at G2 and proceed into mitosis with fatal consequences (Enoch et al. 1992). Thus, if dna2⫹ is required
for DNA replication, the introduction of the checkpoint
mutation, hus1-14, into cells containing the dna2-C2 mutation should allow the double mutant cells to enter
mitosis, leading to catastrophic events at the nonpermissive temperature. Indeed, a significant percentage
(⬎11%) of the hus1-14 dna2-C2 double mutant cells
(HK12, Table 1) underwent aberrant mitosis when
shifted to the nonpermissive temperature (37⬚), producing anucleated cells or progeny cells in which DNA
was distributed unevenly (Figure 4A, right). In contrast,
such aberrant mitosis was not detected in control cells
containing the hus1-14 mutation alone (Figure 4A, left)
Role of Dna2 in Okazaki Fragment Metabolism
or a dna2-C2 single mutant, which arrested as shown in
Figure 2. The same catastrophic result was obtained
when HK13 cells (Table 1) containing both dna2-C2
and rhp9::ura4⫹ were examined (not shown). The rhp9⫹
gene, a fission yeast homolog of S. cerevisiae RAD9, is
required for the DNA damage checkpoint, but not for
the replication checkpoint (Weinert and Hartwell
1988; Willson et al. 1997). These results suggest that
the dna2-C2 mutant at 37⬚ most likely results in the
incomplete replication of chromosomal DNA, which
probably generates damaged DNA structures recognized by the DNA damage checkpoint. This suggests
that nicks or ssDNA regions are present in the newly
replicated DNA in the absence of dna2⫹. Therefore, we
conclude that the dna2⫹ gene has an essential function
in DNA replication at a stage leading to the completion
of duplex DNA synthesis.
Loss of dna2ⴙ function causes qualitatively incomplete chromosome replication: The results described
above indicate that the absence of dna2⫹ function leads
to a defect in DNA replication. To further confirm this,
we analyzed the structure of S. pombe chromosomes using pulsed-field gel electrophoresis (PFGE). As shown
in Figure 4B, chromosomes from wild-type cells entered
the gel and were separated into three chromosomes
irrespective of the incubation temperature (Figure 4B,
lanes 1–4). However, chromosomes isolated from dna2C2 mutant cells (HK10) that were incubated ⬎4 hr at
the nonpermissive temperature failed to yield separated
chromosomes (Figure 4B, lanes 7 and 8). Interestingly,
the low molecular weight smear of DNA observed in
the dna2-C2 mutant was similar to that found in cdc17K42 cells (HK14) whose wild-type protein, DNA ligase
I, functions in the maturation of Okazaki fragments
(Johnston et al. 1986; Waga et al. 1994). The fragmented DNA in both cases accumulated at the bottom
of the gel (Figure 4B). In addition, similarly fragmented
DNA was also observed in cdc24 mutants whose growth
defect was suppressed by dna2⫹ (Gould et al. 1998).
The smear of DNA may result from a qualitative defect
of DNA replication such as pausing of replication forks
exposing frail single-stranded DNA or generation of
premature Okazaki fragments that have nicks or an unprocessed flap structure. Thus, the resulting DNA becomes prone to breakage by nuclease attack or fragile
during experimental manipulation. It is also worthwhile
to mention that the S. cerevisiae dna2-1 mutant synthesized only low molecular weight DNA at the nonpermissive temperature in metabolic labeling studies, suggesting a defect similar to that of the S. pombe dna2-C2
mutant (Budd et al. 1995). These results, as well as those
described above, indicate that the dna2-C2 mutation
has a defect in DNA replication despite its ability to
synthesize bulk DNA.
Isolation of multicopy suppressors for the dna2-C2 ts
mutant: An S. pombe genomic DNA library in the pUR19
vector was screened for genes that could rescue the
1061
Figure 4.—Loss of dna2⫹ function triggers checkpoint control and results in chromosome breakage. (A) The control
strain (hus1-14) or the double mutant HK12 strain containing
hus1-14 dna2-C2 was grown at 25⬚ and shifted to 37⬚ for 8 hr.
The cells were then stained with DAPI and examined under
a microscope. Note that cells on the right were enlarged to
observe the “cut” phenotypes (arrows) more closely. The double mutant cells contained heavily fragmented DNAs, which
appear as speckled spots or unevenly distributed DNA. (B)
Wild-type, HK10 (dna2-C2), and HK14 (cdc17-K42) cells grown
in EMM supplemented with leucine and uracil at 25⬚ were
shifted to 37⬚. The cells were further incubated for 0 (lanes
1, 5, and 9), 2 (lanes 2, 6, and 10), 4 (lanes 3, 7, and 11),
and 8 (lanes 4, 8, and 12) hr at 37⬚. Agarose plugs were
prepared from these cells and the chromosomes were resolved
by pulsed-field gel electrophoresis (PFGE) as described
(Gould et al. 1998). Each plug contained 108 cells and PFGE
was carried out in 0.6% agarose in a CHEF-DR III apparatus
(Bio-Rad, Richmond, CA) for 72 hr in 0.5⫻ TAE buffer at 1.5
V/cm with a switch time of 30 min and 120⬚ of included angle.
In the case where the hydroxyurea arrest control was carried
out (indicated as HU; lane 13), 12 mm (final concentration)
was added to wild-type cultures grown at 25⬚ and cells were
further incubated for 4 hr at 30⬚ prior to preparation of the
agarose plug.
temperature sensitivity of the dna2-C2 mutant using the
procedures described in materials and methods. The
screening of 6.2 ⫻ 105 HK10 cells transformed with the
genomic library yielded 47 independent transformants
that grew at the restrictive temperature. Among these
candidate suppressors, 42 clones (ⵑ90%) contained
wild-type dna2⫹ as expected, and five clones contained
1062
H.-Y. Kang et al.
Figure 5.—Temperature sensitivity of dna2 mutants is rescued
by multicopy or overexpression of
genes involved in Okazaki fragment elongation and maturation.
(A) The mutant cells (HK10) carrying the dna2-C2 allele were
transformed with either vector
(pUR19) alone or pUR19 containing dna2⫹, cdc27⫹, or cdc17⫹.
Aliquots containing 104, 103, 102,
and 10 transformed cells were spotted onto EMM plates containing leucine at the nonpermissive temperature of 34⬚. (B) The
dna2-C2 mutant cells harboring vector (pREP3X) alone, pREP1-dna2⫹, pREP3X-cdc1⫹, or pREP41X-rad2⫹ under control of the
nmt1 promoter were spotted (104, 103, 102, and 10 cells) and grown at 34⬚ in the presence (⫹thi) or absence (⫺thi) of thiamine
on EMM plates containing uracil. Note that the mutants containing dna2⫹ grow faster in the presence of thiamine (repressed
condition) than in the absence of thiamine because overexpression of dna2⫹ caused growth retardation (Formosa and Nittis
1999; Parenteau and Wellinger 1999).
potential extragenic suppressors. Three clones had the
complete sequence of the cdc17⫹ gene encoding DNA
ligase I (Johnston et al. 1986) in common. Two other
clones contained both the complete sequence of the
putative S. pombe fas gene encoding the folic acid synthesis protein (Volpe et al. 1992) and the 5⬘ two-thirds of
the cdc27⫹ gene, a 54-kD subunit of the pol ␦ complex
(MacNeill et al. 1996). The latter two clones were analyzed further to confirm which gene was responsible for
suppression. We found that cdc27⫹ alone was sufficient
and necessary for the suppression of dna2-C2 mutation
(not shown). The growth of the dna2-C2 mutant containing either cdc17⫹ or cdc27⫹ (the 5⬘ two-thirds partial
sequence) as a multicopy suppressor is shown in Figure
5A. In addition, the dna2-C1 mutation was also suppressed equally well by either cdc17⫹ or cdc27⫹ (not
shown), suggesting that the defects associated with dna2C1 and dna2-C2 are similar.
Since cdc27⫹ and cdc17⫹ genes are required for the
elongation and maturation, respectively, of Okazaki
fragments (Ishimi et al. 1988; Goulian et al. 1990; Waga
and Stillman 1994; Waga et al. 1994) and can suppress
the defect observed with the two dna2 ts mutations, it
is most likely that dna2⫹ functions in the Okazaki fragment metabolism. For this reason, we investigated
whether other genes involved in Okazaki fragment metabolism also suppressed the temperature sensitivity of
the dna2-C2 mutation. We constructed plasmids that
contained pol3⫹, cdc1⫹, or cdm1⫹ (pol ␦ subunits) or
rad2⫹ (an S. pombe homolog of RAD27) under the control of the nmt1 promoter (Forsburg 1993; Maundrell
1993). The wild-type dna2⫹ rescued the temperaturesensitive lethality regardless of the presence or absence
of thiamine (Figure 5B). This is due to the high basal
level activity of nmt1 promoter in pREP1 (Maundrell
1993), which is sufficient to complement the dna2-C2
ts mutation. The dna2-C2 mutant cells did not grow at all
when the expression of cdc1⫹ or rad2⫹ was not induced
(Figure 5B). The induction of cdc1⫹ in the absence
of thiamine allowed the dna2-C2 ts mutants to grow
efficiently at the restrictive temperature (Figure 5B). In
contrast, induction of the other pol ␦ subunits sup-
pressed the growth defect of dna2-C2 weakly (cdm1⫹,
encoding the 22-kD subunit of pol ␦) or not at all (pol3⫹,
encoding the catalytic subunit of pol ␦; not shown).
The rad2⫹ gene rescued the dna2-C2 defect when its
expression was induced in the absence of thiamine (Figure 5B). These results demonstrate that the genes involved in the Okazaki fragment elongation or maturation genetically interact with dna2⫹.
The dna2-C2 mutation is synthetically lethal with cdc17K42, rad2::ura4ⴙ, and cdc24-M38: The data presented
above support a role for dna2⫹ in the metabolism of
Okazaki fragments. To further strengthen this conclusion, we examined whether the defect of dna2-C2 can
be exaggerated when combined with mutant alleles of
genes such as cdc17-K42 (DNA ligase I) and rad2::ura4⫹
(Murray et al. 1994). Tetrads obtained from the cross
of dna2-C2 mutant cells (HK10) with either the cdc17K42 (HK14) or rad2::ura4⫹ (HK15) mutant were dissected, and spores were grown at the permissive temperature of 25⬚ (Table 2). In each case, a high proportion of
dead spores was observed (Table 2; 29 and 21% inviable
spores from 21 tetrads of HK10 ⫻ HK14 and 14 tetrads
of HK10 ⫻ HK15 crosses, respectively). The percentage
of dead spores from the two crosses was close to the
expected percentage of dead spores (25%), assuming
the combination of the two unlinked mutations is lethal.
The dna2⫹ gene is on chromosome 2 and thus not
linked to cdc17⫹, cdc24⫹, and rad2⫹, which are on chromosome 1. In test crosses between dna2-C2 and cdc17K42 or between dna2-C2 and rad2::ura4⫹, no double
mutants were detected among the growing spores, confirming that dna2-C2 is synthetically lethal when combined with cdc17-K42 or rad2::ura4⫹ (Table 2).
We also examined the genetic interaction between
dna2⫹ and cdc24⫹, a novel replication gene of fission
yeast essential for chromosome integrity (Gould et al.
1998), to determine whether these two genes are synthetically lethal. For this purpose, we crossed dna2-C2
(HK10) and cdc24-M38 (HK16) mutant strains and analyzed the resulting tetrads. Among 15 tetrads dissected,
25% of the total spores obtained were inviable at 25⬚
(Table 2) and no double mutant was found, suggesting
Role of Dna2 in Okazaki Fragment Metabolism
1063
TABLE 2
Mutations caused synthetic lethality when combined with dna2-C2
dna2-C2
combined with
cdc17-K42
rad2::ura4⫹
cdc24-M38
Tetrad typesa
PD
TT
NPD
Total
spores
Viable
sporesb
Inviable
spores (%)
4
5
3
10
6
9
7
3
3
84
56
60
60
44
45
29
21
25
The dna2-C2 mutant was crossed with cells carrying cdc17-k42, rad2::ura4⫹, or cdc24-M38 and resulting tetrads
were dissected and grown at 25⬚. PD, parental ditype; TT, tetratype; NPD, nonparental ditype.
a
The tetrad types were determined by examining the phenotype of each spore and we excluded from the
analyses the tetrads containing spores that did not germinate. Briefly, in PD tetrads obtained from the cross
of dna2-C2 ⫻ cdc17-K42, all the progeny spores were viable and showed a temperature-sensitive (ts) phenotype.
In NPD tetrads, two spores were wild type (temperature tolerant phenotype) and two spores were inviable
although they germinated under microscopic examination. In TT tetrads, one out of four spores was inviable
and the remaining three viable spores gave a 2:1 segregation of ts:wild type. The absence of double ts mutations
in viable spores was confirmed by backcrossing ts spores with wild-type cells. The backcross always gave rise to
a 2:2 segregation of ts:wild type. The cross of dna2-C2 ⫻ cdc24-M38 or dna2-C2 ⫻ rad2::ura4⫹ was similarly
analyzed.
b
The viability of spores from control crosses was normally ⬎89%.
a synthetic lethal interaction between the two genes.
Fifteen tetrads from crossing of dna2-C2 with cdc27-P11
(MacNeill et al. 1996) were also analyzed. Unlike the
others above, dna2-C2 cdc27-P11 double mutants were
recovered at the expected one-in-four frequency and
viable at both 25⬚ (permissive temperature) and 30⬚
(semipermissive temperature). The failure to observe
a synthetic lethal interaction between the two mutant
alleles may be due to allele specificity. If, for example,
a specific physical interaction is important for viability,
only those mutant alleles that do not allow the physical
interaction would cause the double mutants to be inviable. The synthetic lethality of dna2-C2 when combined
with mutant alleles of several genes known to function in
elongation or maturation of Okazaki fragments suggests
that dna2⫹ is involved in DNA replication, especially at
the stage of Okazaki fragment metabolism.
S. pombe Dna2 interacts with Cdc24, Cdc1, and Rad2
in the yeast two-hybrid system: Since we confirmed the
genetic interactions of dna2⫹ with cdc24⫹, rad2⫹, cdc27⫹,
cdc1⫹, and cdc17⫹, we decided to investigate the physical
interactions between dna2⫹ and those genes using the S.
cerevisiae two-hybrid assay. We constructed a bait plasmid
(pGBT9-dna2⫹) containing dna2⫹ fused to the GAL4
DNA-binding domain (BD) in pGBT9. The cdc24⫹,
rad2⫹, cdc27⫹, cdc1⫹, and cdc17⫹ genes were fused to the
GAL4 activation domain (AD) in pGAD424 or pACT2 to
prepare prey plasmids (pGAD424-cdc24⫹, pACT2-rad2⫹,
pGAD424-cdc27⫹, pGAD424-cdc1⫹, and pGAD424cdc17⫹, respectively) as described in materials and
methods. The plasmid pGBT-dna2⫹ alone or in combination with pGAD424 or each plasmid expressing a
prey protein did not activate transcription of reporter
genes (Table 3 and not shown). When pGBT9-dna2⫹
(BD fusion) and pGAD424-cdc24⫹ (AD fusion) were
cotransformed, a high level of ␤-galactosidase activity
was detected (Table 3). Consistent with this, cells co-
transformed with pGBT9-dna2⫹ and pGAD424-cdc24⫹
turned blue within 1 hr when assayed for ␤-galactosidase
in filter assays (see materials and methods), whereas
control cells hardly turned blue even after 36 hr. This
result indicates a strong interaction between S. pombe
Dna2 and Cdc24. We observed that the reciprocal interaction using pGBT9-cdc24⫹ (BD fusion) and pGAD424dna2⫹ (AD fusion) was weaker, but still significant (not
shown). When pGBT9-dna2⫹ was cotransformed with
either pGAD424-cdc1⫹ or pACT2-rad2⫹ (AD fusions),
reduced levels of ␤-galactosidase activity were detected
(Table 3). Although these activities were relatively low,
they were reproducibly ⵑ10-fold higher than controls
with either pGBT9-dna2⫹, pGAD424-cdc1⫹, or pACT2rad2⫹ alone (Table 3 and not shown). This result suggests weak, but meaningful, interaction between the two
proteins. In keeping with this, cells cotransformed with
pGBT9-dna2⫹/pGAD424-cdc1⫹ and pGBT9-dna2⫹/
pACT2-rad2⫹ turned blue within 8 hr in filter assays.
In contrast, cells containing pGBT9-dna2⫹, pGAD424cdc1⫹, or pACT2-rad2⫹ each alone did not develop blue
color at ⬎36 hr. The reciprocal combinations failed to
lead to detectable ␤-galactosidase activity (not shown),
suggesting orientation-specific interactions in the twohybrid assay. When pGBT9-dna2⫹ was cotransformed
with either pGAD424-cdc27⫹ or pGAD424-cdc17⫹, the
resulting transformants failed to show ␤-galactosidase
activity above background levels (Table 3). However,
the cotransformed cells developed a pale blue color
after prolonged incubation (⬎18 hr) in filter assays,
which are ⵑ106 times more sensitive than the liquid assay
(Table 3). These observations were highly reproducible
and the control plasmid alone did not develop blue
color even after ⬎36 h of incubation (not shown).
To further confirm the interactions observed above,
we examined the expression of the HIS3 reporter gene
by growing cells in the presence of various concentra-
1064
H.-Y. Kang et al.
TABLE 3
Summary of results from two-hybrid analyses
Interactions
Baita
dna2⫹
dna2⫹
dna2⫹
dna2⫹
dna2⫹
dna2⫹
Preyb
␤-galc
X-gald
3-ATe
Suppressionf
Vector
cdc24⫹
cdc1⫹
cdc27⫹
cdc17⫹
rad2⫹
⫺
38.9
0.30
⫺
⫺
0.38
⫺
⫹⫹⫹
⫹
⫾
⫾
⫹
⫺
⫹⫹
⫹
⫺
⫺
⫹
ND
Yes
Yes
Yes
Yes
a
The dna2⫹ was fused to the GAL4 DNA binding domain
fusion.
b
The genes were fused to the GAL4 activation domain.
c
The interactions were determined by measuring ␤-galactosidase activities in Miller units using ONPG as a substrate
(Miller 1972) and the values given represent the average of
duplicate cultures. Background levels (indicated as ⫺) of
␤-galactosidase activity detected with bait or prey alone were
⬍0.0041 ⫾ 0.004.
d
Interactions were monitored by the appearance of blue
color (⫹⫹⫹, formation of blue color within 1 hr; ⫹, within
6–8 hr; ⫾, ⬎18 hr; ⫺, ⬎36 hr) using the colony lift assay on
filter papers containing X-gal (0.33 mg/ml).
e
The HIS3 reporter gene was used to assess the growth (⫹⫹,
wild-type-like growth; ⫹, moderate growth; ⫺, poor or no
growth) on SD plates containing the highest concentration
(30 mm) of 3-aminotriazole (see also Figure 6).
f
Suppression of dna2-C2 mutants by multicopy suppressors
(cdc27⫹ and cdc17⫹) or by overexpression of the genes under
the control of nmt1 promoter (cdc1⫹ and rad2⫹). ND, not
determined.
tions of 3-aminotriazole (3-AT), which suppresses the
leaky growth of false-positive cells (Mangus et al. 1998).
Cells containing each plasmid were grown to midlog
phase and aliquots were spotted on SD plates containing
increasing concentrations of 3-AT (Figure 6). The cells
containing either vector (pGBT9, Figure 6) or bait alone
(pGBT9-dna2⫹, not shown) failed to grow at high levels
of 3-AT (Figure 6). However, cells containing pGBT9dna2⫹ plus pGAD424-cdc24⫹, pACT2-rad2⫹, or
pGAD424-cdc1⫹ grew efficiently in the presence of up
to 30 mm 3-AT (Figure 6). At 20 mm 3-AT, the growth
difference was indistinguishable in cells expressing
cdc24⫹, rad2⫹, and cdc1⫹, whereas the growth of control
cells containing vector alone was extremely poor. These
results suggest that physical interactions exist between
S. pombe Dna2 and Cdc24, Rad2, and Cdc1. We also
investigated the interaction between the prey proteins,
Cdc24, Rad2, Cdc27, and Cdc17, but did not detect
any significant interaction among these proteins (not
shown), suggesting that S. pombe Dna2 serves as a scaffold
protein capable of interacting simultaneously with several proteins. The interactions observed in the yeast
two-hybrid system are consistent with the abilities of
cdc1⫹, cdc27⫹, cdc17⫹, and rad2⫹ to suppress the temperature lethality of dna2-C2. Two of these genes, cdc27⫹
Figure 6.—The dna2⫹ interacts with cdc24⫹, rad2⫹, and
cdc1⫹ in the yeast two-hybrid system. The budding yeast strain
Y190 harboring pGBT9-dna2⫹ as bait was transformed with
pGAD424 (vector), pGAD424-cdc24⫹, pGAD424-rad2⫹, and
pGAD424-cdc1⫹ and the resulting transformants were examined for their ability to express the HIS3 reporter gene in the
presence of increasing concentrations of 3-aminotriazole (3AT). The cells (104, 103, 102, and 10) were spotted on synthetic
minimal medium lacking l-tryptophan, l-leucine, and l-histidine and containing 3-AT at the concentrations indicated.
and cdc17⫹, marginally interacted with dna2⫹; their interactions were detectable only in filter assays (Table
3). In conclusion, the specific genetic and two-hybrid
interactions of dna2⫹ with genes known (DNA ligase,
Fen-1, and pol ␦) or implicated (Cdc24) in Okazaki
fragment metabolism place dna2⫹ as a protein that plays
a critical role in Okazaki fragment elongation/maturation.
DISCUSSION
In this article, we showed that S. pombe spores or vegetative cells with an inactivated dna2⫹ exhibited a distinct
terminally arrested shape similar to that observed with
the mutant cells defective in DNA replication genes
such as cdc24⫹, cdc27⫹, or pcn1⫹ (Waseem et al. 1992;
MacNeill et al. 1996; Gould et al. 1998). They doubled
their DNA content, however, suggesting that S. pombe
Dna2 is not essential for the initiation of DNA replication or the progression of replication forks as suggested
previously for S. cerevisiae Dna2 (Fiorentino and
Crabtree 1997). Since the precise role of a protein in
a multienzyme process such as DNA replication can be
suggested by the proteins with which it interacts, we
attempted to find those that can interact either genetically or physically with S. pombe Dna2. We concluded
that S. pombe dna2⫹ plays a crucial role in the completion
of DNA synthesis on the basis of the following results: (i)
inactivation of S. pombe Dna2 triggered the replication
checkpoint; (ii) loss of S. pombe dna2⫹ function caused
qualitatively incomplete chromosome replication (chro-
Role of Dna2 in Okazaki Fragment Metabolism
mosomes from the S. pombe dna2 mutant were heavily
fragmented at the nonpermissive temperature); and
(iii) S. pombe Dna2 interacted genetically with Rad2,
DNA ligase I, and two subunits of pol ␦ (Cdc1 and
Cdc27), all of which are involved directly in Okazaki
fragment metabolism (Waga and Stillman 1998).
These findings in vivo are also consistent with the results
from our studies in vitro showing that S. cerevisiae Dna2
is a single-stranded DNA-specific endonuclease that is
well suited to completely remove primer RNA on Okazaki fragments (Bae et al. 1998; S.-H. Bae and Y.-S. Seo,
unpublished results).
We observed an increased sensitivity of dna2-C2 mutants to MMS. Since MMS creates adducts and apurinic
sites, which become single- and double-strand breaks
that result from a failure to replicate past lesions containing 3-methyladenine (Schwartz 1989), the dna2
mutants used in this study most likely lack the ability to
remove damaged DNA prior to replicating the lesions,
suggesting a role for Dna2 in DNA repair. The S. pombe
mutant dna2 alleles that were isolated mapped inside
the helicase domain (Figure 1), in keeping with the
previous observation that MMS-sensitive alleles of S. cerevisiae DNA2 clustered in the helicase domain, and the
site-directed motif I and motif II mutations caused MMS
sensitivity (Formosa and Nittis 1999). This result supports the notion that the helicase function is needed
for some forms of DNA damage repair.
The ability of S. pombe dna2⫹ to interact with two
subunits of pol ␦, Rad2, and DNA ligase I raises the
possibility that Dna2 exists in vivo within a multiprotein
complex. On the basis of the results from yeast twohybrid analyses (Table 3), however, interactions of S.
pombe Dna2 with two subunits (Cdc1 and Cdc27) of pol
␦ may not be strong enough to allow formation of a
stable complex between Dna2 and pol ␦. Other interactions of S. pombe Dna2 with Rad2 and DNA ligase I would
be necessary for Dna2 to be stably tethered to pol ␦.
Under these circumstances, the ability of Rad2 and DNA
ligase I to complex directly with PCNA (Li et al. 1995;
Levin et al. 1997; Montecucco et al. 1998; Gary et al.
1999b) could stabilize further the association of S. pombe
Dna2 with pol ␦, since PCNA itself is tightly coupled to
pol ␦ while DNA synthesis occurs. This would account
for the following discrepancy: we failed to observe any
detectable complex formed between purified recombinant Rad27 and Dna2 of S. cerevisiae (not shown). In
contrast, both Rad27 and S. cerevisiae Dna2 copurified on
an immunoaffinity column, and they were reciprocally
coimmunoprecipitated from crude extracts (Budd and
Campbell 1997). In support of the hypothesis that Dna2
exists in a multiprotein complex, S. cerevisiae Dna2 in
cell extracts eluted from a size-exclusion matrix column
at ⵑ700 kD, although this complex has not been analyzed yet (Formosa and Nittis 1999).
Recent genetic studies on the mutant alleles of rad27
are also in accord with our hypothesis (Gary et al. 1999b).
1065
The growth of mutant cells containing rad27-n (a nuclease-deficient allele of RAD27) was severely inhibited,
whereas the growth of rad27⌬ or rad27-p (defective in
the PCNA-binding site) mutant cells was not (Gary et
al. 1999a,b). The double mutant (rad27-n,p) was not
inhibited. These observations were interpreted as follows. In rad27-n cells, the interaction of Rad27-n with
PCNA allows the mutant protein to occupy its normal
position within a multiprotein complex, which could
hinder the action of secondary or redundant enzymes
capable of processing the Okazaki fragment. In the case
of rad27-n,p or rad27-p, the mutant proteins were not
integrated within the multiprotein complex, hence
allowing access of an alternative enzyme. These observations not only support the idea that processing of Okazaki fragments occurs in the context of a multiprotein
assembly, but also predict the existence of a secondary
processing enzyme that can operate in the absence of
Fen-1 (Rad27). We believe that the enzyme is most likely
Dna2 for the reasons described elsewhere (S.-H. Bae
and Y.-S. Seo, unpublished results).
Dna2 may be regulated by proteins with which it interacts. Such a possibility is supported by significant differences in enzymatic activities noted between the recombinant S. cerevisiae Dna2 purified from insect cells and the
one from S. cerevisiae cell extracts (Budd and Campbell
1995; Bae et al. 1998). Alternatively, Dna2 can alter
enzymatic properties of proteins such as pol ␦ or Fen-1
via protein-protein interaction in order to coordinate
the complicated multienzyme process of Okazaki fragment elongation and maturation. For example, the specific interaction between Dna2 and pol ␦ would lead to
a change in the ability of pol ␦ to displace the 5⬘-end
region of the preexisting Okazaki fragments. We showed
that a flap structure generated by displacement DNA
synthesis by pol ␦ was rapidly removed by S. cerevisiae
Dna2 (S.-H. Bae and Y.-S. Seo, unpublished results).
Considering that pol ␦ is capable of displacement DNA
synthesis up to 274 bp, longer than the size (100–150
nucleotides) of Okazaki fragments (Mossi et al. 1998),
one possible consequence of Dna2-pol ␦ interaction
would be the timely disassembly of the pol ␦ complex
when no further displacement is required. At present,
the roles played by Dna2 in the context of a multienzyme
complex are highly conjectural and rigorous biochemical studies are needed to define any role of Dna2 in
this regard. Recently, an additional role for Dna2 has
been suggested by the observation that S. cerevisiae Dna2
interacts with POL1 and CTF4, which encode the DNA
polymerase ␣ catalytic subunit and an associated protein, respectively (Formosa and Nittis 1999). This suggests that Dna2 may also act in a process that involves
pol ␣ in addition to the roles suggested above. Although
pol ␣-primase plays an essential role in Okazaki fragment initiation (Tsurimoto et al. 1990; Waga and
Stillman 1994; Waga et al. 1994), the precise link between Dna2, pol ␦, and pol ␣ is not clearly understood.
1066
H.-Y. Kang et al.
There are a couple of differences noted in the DNA
replication apparatus between fission yeast and budding
yeast. For instance, S. pombe pol ␦ has one additional
subunit, Cdm1, which has no structural counterpart in
S. cerevisiae (MacNeill et al. 1996; Zuo et al. 1997; Gerik
et al. 1998). Another example is that S. cerevisiae lacks
the structural equivalent of S. pombe cdc24⫹ (Gould et
al. 1998). The synthetic lethality of dna2-C2 cdc24-M38
and the two-hybrid interaction of S. pombe dna2⫹ with
cdc24⫹ suggest that cdc24⫹ could affect the function of
dna2⫹ and, like dna2⫹, it is most likely involved in Okazaki fragment metabolism. At the same time, these findings suggest that the mode of action of S. pombe Dna2
is likely to be different from that of S. cerevisiae Dna2.
It is worthwhile to mention that the N-terminal 405
amino acids could be deleted without loss of any enzymatic activities of S. cerevisiae Dna2 (Bae et al. 1998),
whereas deletion of ⬎105 amino acids from its N terminus resulted in cell death (Budd et al. 1995). These two
observations suggest that the N-terminal region of S.
cerevisiae Dna2 plays an essential regulatory role for Dna2
function, which may be required either to regulate the
enzymatic activities of Dna2 or to serve as a site of protein-protein interaction. The N-terminal amino acid sequences are only weakly conserved among Dna2 homologs and do not contain motifs characteristic of any class
of protein with known function. Therefore, the unique
N-terminal amino acid sequence of each Dna2 protein
may serve as a site of protein-protein interactions specific for its own regulatory protein. In keeping with this
possibility, the cdc24⫹ gene specifically interacted with
the N-terminal half of S. pombe dna2⫹ in the yeast twohybrid assay (not shown). This may provide an explanation for the genetic and biochemical differences observed in the Dna2 proteins of fission yeast and budding
yeast.
We thank Dr. Jerard Hurwitz for critical reading of the manuscript
and comments on the manuscript. We also thank all the members of
our laboratory for their valuable discussions. We are especially grateful
to the members of laboratories of Drs. S. A. MacNeill, Y. Adachi, and
P. A. Fantes in the University of Edinburgh for their kind help during
the initial stage of this work. We thank the following people for
providing strains, plasmids, and genomic DNA libraries: Dr. J. M.
Murray and F. Z. Watts (University of Sussex, UK), Dr. H. Ohkura
(University of Edinburgh, UK), Dr. J. Hurwitz (Sloan-Kettering Institute, USA), Dr. J. Rho (Seoul National University, Korea), and Dr.
H. Yoo (Korea Research Institute of Bioscience and Biotechnology,
Korea). We are greatly indebted to A. Sanderson (University of Edinburgh, UK) for FACScan analyses. This work was supported by a
grant from the Creative Research Initiatives of the Korean Ministry
of Science and Technology given to Y.-S.S.
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