A fission yeast gene encoding a protein that preferentially associates with curved DNA

YEAST VOL. 1 0 883-894 (1994)

A Fission Yeast Gene Encoding a Protein that Preferentially Associates with Curved DNA HISAMI YAMADA, HIDEKI MORI, HIROYUKI MOMOI, YOSHIYUKI NAKAGAWA*, CHIHARU UEGUCHI AND TAKESHI MIZUNOt

Laboratory of Molecular Microbiology, School of Agriculture and *Biological Laboratory, College of General Eduction, Nagoya University, Chikusa-ku, Nagoya 464, Japan

Received 28 September 1993; accepted 27 January 1994

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA-binding gel shift assay in the presence of an excess amount of a non-curved DNA sequence as a competitor. We identified such a protein in S. pornbe. The protein, thus purified, has an apparent molecular weight of 42 000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was suggested that this protein (42 K-protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non-curved DNA sequence with lower affinity. As its putative coding sequence, a 1.9-kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381-amino-acid protein with a calculated molecular mass, 41 597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K-protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.

KEY WORDS - Fission yeast; DNA curvature; gel shift assay; DNA-binding protein; cloning and sequencing.

INTRODUCTION

The results of recent studies have demonstrated the existence of intrinsic (sequence-directed) DNA curvature in free double-stranded DNA segments (for reviews, see Hagerman, 1990; Crothers et al., 1990). Curved DNAs are characterized generally by the periodical arrangement of short dA stretches in approximate phase with the DNA helical repeat (Koo et al., 1986), although other types of DNA sequences are also capable of induc- ing DNA curvature (Bolshoy et al., 1991). Several distinct models for theoretical rationalization of the striking dependence of DNA curvature on the base sequence have been advanced (for a review, see Crothers et al., 1990). The degree of DNA curvature can be experimentally detected and estimated on the basis of the anomalous electrophoretic mobilities of DNA fragments in a polyacrylamide gel (Koo et al., 1986; Diekmann, 1986; Hagerman, 1986).

?Addressee for correspondence.

0 1994 by John Wiley & Sons Ltd CCC 0749-503X/94/070883-12

Although there have been numerous reports of DNA molecules exhibiting intrinsic curvature in a wide variety of naturally occurring DNA sources, their biological significance remains obscure. However, there have also been a number of reports on curved DNAs implying a potential function in crucial biological processes such as replication, recombination and transcription (for a review, see Hagerman, 1990). The implication arising from such studies is that the DNA curvature is an important determinant of both the function and the architecture of a nucleoprotein complex involved in a variety of biological processes (for a review, see Travers, 1989). To gain insight into the functional importance of the DNA curvature, we systematically searched for curved DNA segments in total genomic DNA sources, such as those from a bacterium (Escheri- chia coli) and a yeast (Saccharomyces cerevisiae) (Mizuno, 1987; Mizuno and Itoh, 1988; Tanaka et al., 1991). As a complementary approach, we searched for curved DNA-associated proteins in E. coli (Yamada et al., 1990). Such an E. coli

884 H. YAMADA ET AL.

protein was indeed isolated, and shown to be the so-called E. coli histone-like (nucleoid) protein, H-NS (Bracco et al., 1989; Yamada et al., 1990). In this study, we searched for curved DNA- binding proteins in a eukaryotic microorganism, Schizosaccharomyces pombe (fission yeast). We purified and characterized a candidate, and its coding gene was cloned and sequenced.

MATERIALS AND METHODS Materials

Restriction endonucleases, bacteriophage T4 DNA ligase and polymerase, the Klenow fragment of DNA polymerase, a DNA kilo-deletion kit and a DNA sequencing kit were obtained from Takara Shuzo Co. or Toyobo Co. Zymolyase-lOOT was from Seikagaku Kogyo Co. [CZ-~~PI~CTP (3000 Cil mmol) and [Y-~~PIATP (5000 Cilmmol) were pur- chased from Amersham International. Degenerate oligonucleotides (5'-dTTRTAYTTRTTNACNG TYTC and 5'-dCCNGCNATYTTRTAYTTRTT) were synthesized with an automated DNA syn- thesizer (Applied Biosystems, Center for Gene Research, Nagoya University).

Bacterial and yeast strains E. coli K-12 JM83 [recA araD Alac-proAB rpsL

thi Q80 (IacZAMlS)] was mainly used as a host strain. Bacteria were grown in Luria broth (10 g/1 tryptone, 5g/l yeast extract, 5g/l NaCl), or on plates containing 1.5% agar. When required, ampi- cillin (50 pg/ml) was added. S. pombe strains used in this study are: 972 h - [wild-type], HM123 [h- leul], TP4-5A [h- leul ural ade6-M210] and TP4-1D [h+ leul ural his2 ade6-M216] (all generous gifts of M. Yanagida). These strains were mainly grown in YPD medium (Moreno et al., 1991).

DNA-binding gel shift assay The DNA-binding gel shift assay was carried

out in 20 p1 reaction mixtures containing 3 2 ~ -

labeled DNAs and proteins in 10 mM-Tris-HC1 (PH 7.5), 1 mM-EDTA, 80 Mm-NaC1, 10 m ~ - 2 - mercaptoethanol, 4% glycerol. Each reaction mix- ture was incubated for 20 min at room temperature, and then immediately loaded onto a polyacrylamide gel containing 6% acrylamide, 0.1% bis-acrylamide, 40 mM-Tris-HC1 (pH 7*4), 5 mM-sodium-acetate and 1 mM-EDTA. After electrophoresis, the gels were subjected to autoradi-

ography. When necessary, densitometric quantification of the autoradiograms was carried out with a dual-wavelength scanner (Shimazu, CS- 9000).

Preparation of S. pombe cell extract and puriJication of the 42 K-protein

S. pombe strain 972 h - was grown in YPD medium containing 10% yeast extract, 2% polypeptone, 2% glucose (pH 5.5) and harvested at the late-logarithmic growth phase. Two hundred ml of a buffer containing 1 M-sorbitol, 50 mM-Tris- HCl (pH 7.4), 10 mM-MgCl,, 3 mwdithiothreitol, 1 mM-phenylmethylsulfonyl fluoride, 2-5 pg/ml leupeptin, 2.5 pg/ml pepstatin-A was added to 100 g of cells. The suspension was incubated in the presence of 0.75 mg/ml Zymolyase 100-T at 37°C for 60 min to prepare protoplasts. The protoplasts were recovered by centrifugation (2700 x g , for 5 rnin), and suspended in a buffer (100 ml) containing 10 mM-HEPES (pH 7.8), 15 mM-NaC1, 5 mM- MgCl,, 0.1 mM-EDTA, 3 mM-dithiothreitol and the protease inhibitors described above. The sample was homogenized with Dounce homogenizer (20 strokes), and then centrifuged at 10 000 x g for 10 min. The pellet was suspended with the same buffer as described above, and then NaCl was added at a final concentration of 0.5 M. The sample was kept on ice for 20 min, and centrifuged at 100 000 x g for 60 min. The super- natant was used as a crude nuclear extract of S. pombe. The crude nuclear extract was dialysed against a buffer containing 10 mM-potassium phosphate (pH 6-9) and 2 m~-2-mercaptoethanol. The dialysed sample was applied to a phosphocellulose P11 (Whatman) column (1.9 x 50 cm) previously equilibrated with 10 mM-potassium phosphate (pH 6.9), and then proteins were eluted with a 700 ml linear potassium phosphate gradient, from 10 to 700mM, at the flow rate of 30mYh. Fractions containing the 42 K-protein were pooled ((raction f i n Figure 2). This fraction was dialysed against 10 mM-potassium phosphate (pH 7-59, 1 mM- EDTA, 2 rnw-2-mercaptoethanol. The dialysed sample was applied to a heparin-Sepharose CLdB (Pharmacia) column (0-9 X 13 cm) previously equilibrated with the same buffer, and then proteins were eluted with a 240 ml linear NaCl gradient, from 0 to 800 mM, at a flow rate of 15 mlh. Fractions containing the 42 K-protein were further subjected to a FPLC column chromatography system (LKB) equipped with a Mono-S COhmn.

DNA-BINDING PROTEIN IN FISSION YEAST 885

The conditions of the column chromatography were as follows: buffer, 20 mM-sodium acetate (pH 5.3), 1 mM-EDTA, 2 m~-2-mercaptoethanok gradient, 0-600 mM-NaC1; flow rate, 0.75 mumin. The 42 K-protein was further purified with Mono-Q column. The conditions of this final column chromatography were as follows: buffer, 10 mM-potassium phosphate (pH 8.2), 1 mM- EDTA, 2 m~-2-mercaptoethanol; gradient, 0-500 mM-NaC1; flow rate, 0.75 mumin. The 42 K- protein-containing fractions, which were essentially free from other proteins, were combined and used as the purified 42 K-protein. Amino acid sequencing was performed with an automated amino acid sequencer (Applied Biosystems, Model-447A).

Cloning and DNA sequencing The 14.7 kb DNA fragment in an isolated clone

of S. pombe genomic library was appropriately shortened and subcloned onto pUC19. One of the resultant plasmids, carrying the HindIII-EcoRI region, was used for DNA sequencing (see Figure 7). Sequencing of double-stranded DNAs on pUC19 was performed according to the method of Chen and Seeburg (1985) using the chain- termination method of Sanger et al. (1977) with a sequencing kit. Generation of successively shortened subclones for sequencing was performed using a kilo-sequence deletion kit.

Plasmids for overproduction of the 42 K-protein and tetrad analysis of its coding gene

The 1.4 kb Sun- VspI fragment encompassing the 42 K-protein open reading frame (ORF) was isolated, and then blunt-ended with T4 DNA polymerase and inserted into an E. coli overexpres- sion vector (pUSI-2) (Shibui et al., 1988) at its KpnI site, which had previously been blunt-ended (see Figure 7). The resultant plasmid was designated pOP42. The 4-9 kb BamHI-Hind111 fragment was inserted into the BamHIIHincII sites of plasmid pUC118. Note that the HindIII site had previously been blunt-ended with T4 DNA polymerase. To disrupt the 42 K-protein coding sequence in the resultant plasmid (pCUY6) gene, the 1.8-kb HindIII fragment carrying uru4+ gene was inserted into the unique SalI site of pCUY6 (see Figure 6). The ura4+ gene was isolated from plasmid pPuru4+ (generous gift of M. Yanagida). The tetrad analysis was carried out by the conven- tional method (Moreno et al., 1991).

Screening of genomic and cDNA libraries An S. pombe genomic library (Sau3AI partial

digests cloned onto an E. coli vector, pDB248', generous gift of M. Yanagida) was screened with radiolabeled oligonucleotide probes according to the method of Maniatis et al. (1982). A cDNA library of S. pombe (1ZAPII-vector, generous gift of M. Yanagida) was screened with radiolabeled DNA probes according to the method of Singh et al. (1987).

Isolation of S. pombe nuclear fraction S. pombe strain HM123 h - leu1 was grown in

200 ml of EMM medium containing 0.5% glucose and 50 pg/ml leucine (Moreno et al., 1991). First of all, protoplast preparation was prepared by essentially the same procedure as described above. The protoplasts were homogenized in a Ficoll buffer (1 ml) containing 15% Ficoll 400 (Pharmacia), 20 mwpotassium phosphate (pH 6-5), 0.1 mM- CaCl,, 1 mM-phenylmethylsulfonyl fluoride, 2-5 vg/ml leupeptin, 2.5 pg/ml pepstatin-A, by Dounce homogenizer (10 strokes). The homoge- nate was directly layered on a 1540% linear Percoll gradient (Pharmacia), which was made in a buffer containing 20 mM-potassium phosphate (pH 6.5), 1 M-sorbitol, 0-1 mM-CaCl,. The nuclear fraction, migrating at the middle of the gradient after centrifugation (2500 x g, for 30 min), was collected, and then diluted with 15% Ficoll solu- tion (4 times by volume). The purified nuclear fraction was collected by centrifugation (25 000 x g, for 10 min), and then suspended in a buffer containing 1 M-sorbitol, 50 mM-Tris-HC1 (PH 74), 10 mM-MgC1,.

Fluorescence microscopy Fluorescence microscopic observation of the

purified nuclear fraction was carried out essentially as described previously by Hagan and Hyams (1988). Briefly, the nuclear fraction was fixed with 3% formaldehyde for 60 min at 33°C. The sample was treated with 4',6-diamidino-2-phenylindol (DAPI). The sample was air dried on poly-L- lysine ( I mg/ml)-coated coverslips, and then in- verted onto glycerol-mounting medium containing 1 mg/ml p-phenylenediamine.

Western blot analysis Proteins were first subjected to sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS- PAGE; Laemmli, 1970). The proteins in the gel


S10-15: [GGCAAAAACG] X 15

EcoRl 4 Hlndlll

SI 5-1 2: [CCGGCAAAAACGGGC 1 x 12 Figure 1. Synthetic curved and non-curved sequence used in this study. S10-15 (200 bp) contains tandem repeats of the indicated 10 mer ( x 15) flanked by the EcoRI-Hind111 polylinker sequence of pUC19. S15-12 (233 bp) contains tandem repeats of indicated 15 mer ( x 12). The DNA duplex, generated from the synthetic oligonucleotides 5'- dGGCAAAAACG and 5'-dCCCGTTITTG, was ligated to obtain a multimer of it. Similarly, another DNA duplex, generated from 5'-dCCGGCAAAAACGGGC and 5'- dGGGCCCGTTTTTGCC, was also ligated. These multimers, thus prepared respectively, were blunt-ended by treatment with the Klenow fragment and then cloned into the unit SmaI site of puc19.

were transferred to a nitrocellulose filter. The filter was treated with mouse polyclonal antiserum raised against the 42 K-protein, and then further treated with alkaline phosphatase-coupled goat anti-mouse immunoglobulin G to detect cross- reactive proteins.

RESULTS AND DISCUSSION Rationale of the experimental design for isolating proteins that preferentially bind to curved DNA

As shown in Figure 1, two types of synthetic DNA sequences, both of which contain consecutive dA, stretches, were designed and cloned previously into plasmid pUC19 (Yamada et al., 1990). One of the synthetic sequences, designated S1&15 (200-bp EcoRI-Hind111 fragment), contains tandem repeat (15 times) of a 10 mer (5'- dGGCAAAAACG), in which dA, stretches were designed to be phased at 10-bp intervals. The other sequence, designated S15-12 (233-bp EcoRI- Hind111 fragment), contains tandem repeat (12 times) of a 15 mer (5'-dCCGCAAAAACGGC), in which dA, stretches were designed to be phased at 15-bp intervals. Short homopolymeric runs of dA, when periodically repeated in approximate phase with the DNA helical repeat, can confer a significant degree of curvature on an isolated DNA molecule (Koo et al., 1986). According to the current concept of DNA curvature, S10-15 must have an overall and strong curvature, as was

indeed demonstrated previously by its extremely anomalous gel electrophoretic mobility (Yamada et al., 1990). On the other hand, S15-12 shows no detectable signs of overall DNA curvature, as confirmed by the same analysis (and see Koo et al., 1986). In this study, we attempted to find S. pombe proteins which preferentially associate with S 10- 15, even in the presence of a large excess amount of S15-12 as a competitor, by means of gel electrophoretic DNA-binding assay (gel shift assay). Since the two sequences had nearly the same sequence motif, including consecutive dA, stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature, and other sequence-specific DNA-binding proteins (e.g. dAn-binding protein) being excluded.

PurlJication of an S. pombe protein which binds to a synthetic curved DNA with high aflnity

A crude nuclear extract of S. pombe was fractionated by phosphocellulose column chromatography (Figure 2A). The fractions eluted from the column were pooled into seven fractions (a to g). Each protein pool was then subjected to a gel shift assay (Figure 2B). Several fractions indicated the presence of a protein(s) which may preferentially bind to S10-15. In this study, we focused our attention on a particular fraction (f) (we have not yet succeeded in purifying any protein from fractions e and g, which also exhibit the relevant property). Proteins in fraction f were further fractionated by successive heparin-Sepharose, Mono-S and Mono-Q column chromatography, with concomitant monitoring by means of a gel shift assay (Figure 3). A protein with an approximate molecular weight of 42 K, which binds to S10-15 with strong affinity, was purified to near homogeneity (lane 6).

The 42 K-protein preferentially binds to a curved DNA

The DNA-binding property of the purified 42 K-protein was examined more closely. First of all, Figure 4 shows the preferential association of the 42 K-protein with S10-15, as the binding was easily competed for by cold S10-15, but not by a large excess of cold S15-12 (up to 1000 times by weight), although the relative mobility of the 42 K- proteidDNA complex on the gel was somehow affected. To characterize further the preferential binding of the 42 K-protein to S10-15, 32P-labeled


I " 1

0.0

Fraction Number

B F a b c d e f g

Free-DNA)

Figure 2. Fractionation of proteins which preferentially bind to a synthetic curved DNA. (A) A crude nuclear-protein extract from S. pombe was fractionated on a phosphocellulose column with linear gradient elution (1 0-700 mM-KH,PO,-K,HPO,). Fractions, indicated by horizontal bars, were pooled. These fractions, denoted by small letters (a-g), were used. (B) Aliquots (15 pg) of the fractions were examined by means of a gel shift assay for the presence of proteins binding to 32P-labeled SIO-15 in the presence of an excess amount of non-labeled S15-12 (50 times by weight) as a competitor. Free 32P-labeled S10-15 was run in lane F.

S10-15 was incubated with increasing concen- passes the multi-cloning sites. This nucleotide trations of the 42 K-protein in the presence of cold S15-12 as a competitor (Figure 5). On the other hand, 32P-labelled S15-12 was also incubated in the presence of cold S10-15 under the same conditions (Figure 5). The samples were then analysed by the gel shift assay, and the results are presented quan- titatively. Highly preferential binding of the 42 K- protein to S10-15 was observed. At higher protein to DNA ratios, however, a binding of the 42 K- protein to S15-12 appeared to occur, suggesting that this protein binds to a non-curved DNA, albeit with lower affinity. These results suggested that the 42 K-protein preferentially binds to a curved DNA, relative to a non-curved one.

The observed preferential binding of the 42 K- protein to the curved DNA was further characterized in a different way, as shown in Figure 6. In this case, a naturally occurring DNA segment from plasmid pUC19 was used as a negative reference. This pUC19 fragment corresponds to the 141-bp HindIII-PvuII region which encom-

sequence shows no signs of DNA curvature, as judged from its mobility on polyacrylamide gel (data not shown). A set of DNA fragments, containing different numbers of the 10 mer sequence (5'-dGGCAAAAACG) inserted at the HincII site of the pUC19 sequence, were prepared (Figure 6). The 42 K-protein exhibited significant affinity with the control pUC19 fragment in the absence of a competitor (i.e. N=O), exhibiting a single (but broad) retarded band on the gel. However, this protein clearly showed preferential binding to the DNA fragments with increasing numbers of the insert (for example, N=5 and N=6) , even in the presence of a large excess of the competitor. Considering that the set of target DNA fragments exhibited various degrees of DNA curvature, due to the different numbers of the insert, this suggests that the DNA-binding ability of the 42 K-protein is dependent upon the degree of DNA curvature. From these results of different DNA-binding assays (Figures '4-6), it was suggested that the


1 2 3 4 5 6 Competitor SIO-15 S15-12

94 - '

67

43 -

30 - Free DNA *

Figure 3. Purification of the 42 K-protein. The samples and amounts of proteins applied on SDS-PAGE were: lane I , total cell lysate (120pg); lane 2, cytosolic fraction (80pg); lane 3, crude nuclear-protein extract (80 pg); lane 4, proteins from phosphocellulose chromatography (fraction j) (20 pg); lane 5, proteins from heparin-Sepharose chromatography (30 pg); lane 6 , proteins from Mono-Q chromatography (5 pg). Molecular standards are indicated in kDa on the left-hand side of the panel.

42 K-protein associates preferentially with a curved DNA sequence, although it also binds to a non-curved DNA sequence with lower affinity. Based on the result shown in Figure 6 , for example, the 42 K-protein was estimated to associate lo2 times more efficiently with the curved DNA than with the pUC19 fragment, since the complex with the former did not result in release of the DNA fragment even in the presence of a 102-fold excess of the pUC19 fragment.

Cloning of genomic and complementary DNAs encoding the 42 K-protein

We attempted to determine the N-terminal amino acid sequence for the purified 42 K-protein. The N-terminus appeared to be blocked by a modification. Therefore, the protein was partially digested with mild trypsin treatment. A poly- peptide with a slightly lower molecular weight was generated (data not shown). After electro- elution of the tryptic digest from SDS-PAGE, its N-terminal amino acid sequence was determined

0 0 0 0

0 0 0 0 0 0 C U V ) V ) N * r

O X X X ~ X X

Figure 4. DNA-binding properties of the 42 K protein. The Mono-Q-purified 42 K-protein (0.5 pg) was incubated with 32P-labeled SIO-15 (10 ng) in the absence of any competitor DNA (lane 0) and in the presence of increasing amounts of the indicated cold competitor DNAs (either SIO- 15 or S15-12). These samples were subjected to a gel shift assay (see Materials and Methods), followed by autoradiography.

to be EATSETAVDYSLSNPETVNKYKIAGEV. Two types of degenerate oligonucleotide mixtures were prepared, one corresponding to the sequence, ETVNKYK, and the other to VNKYKIAG (see Materials and Methods). A genomic DNA library, prepared from S. pombe in an E. coli plasmid- vector, was first screened with the probe for ETVNKYK, by means of colony-hybridization. A clone carrying a 14.7 kb DNA fragment was identified. This clone was further confirmed by using the probe for VNKYKIAG. The region that hy- bridized with both the probes was narrowed down by subcloning (Figure 7). The 1.9-kb HindIII- VspI region, within which the putative coding sequence in question appeared to be located, was subjected to nucleotide sequencing. The results are shown in Figure 8. In order to assign correctly the ORF in the determined nucleotide sequence, a cDNA library, prepared from S. pombe with the 1ZAPII- vector, was also screened with an appropriate genomic DNA probe (the 1.3-kb HindIII-EcoRI fragment, see Figure 7). A cDNA clone with a poly-A tail was isolated (see Figure 7). This cDNA clone was also subjected to restriction mapping and partial nucleotide sequencing. Taking all the results together, the ORF was unambiguously as- signed to that shown with the nucleotide sequence


[GGCAAAAACG ] x N

2 a m 0 40

20

0 0 .0 0.2 0.4 0.6 0.8 1.0 1.2

42K-Protein ( pg) Figure 5. Concentration dependence of the binding of the 42 K-protein to a synthetic curved DNA. Increasing amounts of the 42 K-protein were incubated with 32P-labeled S10-15 (10 ng) in the presence of an excess amount of cold S15-12 (50 times by weight). Similarly, increasing amounts of the 42 K- protein were incubated with 32P-labeled S15-12 (10 ng) in the presence of an excess amount of cold S10-15 (50 times by weight). These samples were subjected to a gel shift assay, followed by autoradiography. The amounts of protein-bound DNA were determined by densitometric quantification of the autoradiograms.

(Figure 8). The predicted ORF (1 143 bp) has no intron, and was revealed to encode a protein consisting of 381 amino acids with a calculated molecular mass of 41 497 Da. This value is in good agreement with that estimated for the 42 K-protein by means of SDS-PAGE. Furthermore, exactly the same amino acid sequence determined for the tryptic digest of the 42 K-protein was located close to the N-terminal end of the predicted amino acid sequence, being preceded by a short sequence, Met-Ser-Thr-Lys (see Figure 8). This is consistent with the fact that the digest of the 42 K-protein exhibited a molecular weight very close to the original one, and that trypsin should cleave the C-terminal end of Lys or Arg residues.

To confirm this ORF, the 1.4-kb Sun-VspI sequence encompassing the entire ORF was placed under the control of an inducible E. coli expression vector (Figure 7). The total cellular lysate was isolated from E. coli cells carrying this particular

+ 42K-Protein + Competitor (pUC19 Fragment)

Gel shift assay

100

80 n

8

n

m

2 60 z

40 0

20

0

I

1 I\ \ N = 5

0.0 1 .o 2.0 Competitor ( pg) (pUC19 Fragment)

Figure 6. DNA-binding properties of the 42 K-protein. In- creasing numbers of the 10 mer sequence were inserted into the multi-cloning sites (HincII) of the plasmid pUC19, as indicated. Then, the PvuII-Hind111 fragment was isolated and labeled with 3zP. These '*P-labeled DNA fragments (10 ng each) were incubated with the 42 K-protein (0.5 pg) in the absence and presence of increasing amounts of the competitor (the 141-bp PvuII-Hind111 fragment with no insertion). These samples were subjected to a gel shift assay, followed by autoradiography. The amounts of protein-bound DNA were determined by densitometric quantification of the autoradiograms.

plasmid (grown under either non-inducible or inducible conditions). When analysed by SDS- PAGE (Figure 9A), an inducible protein with the same electrophoretic mobility as that of the 42 K- protein was clearly detected in the gel (lane 2). These samples were subjected to Western blot analysis with mouse antiserum raised against the 42 K-protein (Figure 9B). The protein band expressed in E. coli cells cross-reacted with this antiserum (lane 2). From these results, we con- clude that this s. pombe gene does indeed specify the 42 K-protein, which was identified as a curved


sal I vsp I EcoRl Hindlll

Bawl f5 sal I Hindlll EcoRl

I 1 kb

cDNA 1 'An 1

sequencing tac-promoter

E. coli Plasmid pOP42 ---* RBS

ATG Figure 7. Schematic representation of the S. pombe genomic DNA region containing the gene encoding the 42 K-protein. The represented DNA segment was subcloned on plasmid pUC19 from an original 1ZAPII-clone. The 1905-bp HindIII-VspI region, indicated by the arrow, was subjected to nucleotide sequencing. Within this region, a complete ORF was found (see Figure 8), as indicated by the shaded rectangle. A cDNA clone, originating from this particular genomic region, was also isolated and subjected to restriction mapping and nucleotide sequencing. The determined structure for the cDNA clone with a poly-A tail (An) is also shown. The Sun- VspI region, encompassing the 42 K-protein coding sequence, was placed under the control of an E. coli expresison vector @USI-2), containing the tuc promoter, an ideal ribosome binding site (RBS) as well as the ATG initiation codon. Note that the Sun site is located in the initiation codon of the 42 K-protein coding sequence (see Figure 8). The structure of the resulting plasmid (pOP42) is schematically shown. To create a disruption mutant of the 42 K-protein coding region in S. pombe, the uru4+ gene was inserted into the Sun site, as indicated (see Materials and Methods).

DNA-binding protein. It should be noted, however, that the overproduced 42 K-protein in E. coli cells was found to form insoluble inclusion bodies. Thus, so far, we have not succeeded in isolating this protein from E. coli cells as a soluble form. The biochemical properties of the E. coli-expressed 42 K-protein remain to be elucidated, particularly in order to confirm its DNA-binding ability. In this respect, the same sample as analysed in Figure 9 was sub'ected to a South-Western blotting analysis with P-labeled SIO-15 DNA as a probe (data not shown). The preliminary result suggested that the E. coli-expressed 42 K-protein binds the probe DNA.

1

Properties of the predicted amino acid sequence for the 42 K-protein

A computer-aided search was conducted, using the GenBank and Swiss Prot databases for amino acid sequences homologous to that deduced for the 42 K-protein. The predicted protein sequence is not similar to any sequences, including an E. coli curved DNA-binding protein (i.e. H-NS, see Intro- duction). An exception is a hypothetical C. elegans protein. This hypothetical protein has been previ-

ously predicted from a C. elegans cDNA clone (from a cDNA library of 1517 clones), the partial sequence of which was systematically determined and analysed (Waterston et al., 1992). As shown in Figure 10, the 42 K-protein shares 43% sequence identity with this hypothetical C. elegans protein over 140 amino acids (note that only the N-terminal amino acid sequence has been reported for the C. elegans protein). Considering the phylo- genetic distance between S. pombe and C. elegans, this apparent similarity is clearly significant. How- ever, its functional significance is not clear at present. It is also worth mentioning that the 42 K- protein displays no obvious similarity to known DNA-binding motifs, e.g. helix-turn-helix, Zn- finger or b-ZIP.

Localization of the 42 K-protein Since the 42 K-protein was purified from a crude

nuclear extraction, it was assumed to be located in the nucleus. To confirm this assumption, the nuclear fraction was isolated and purified from S. pombe cells (see Materials and Methods). As shown in Figure IIA, this subcellular fraction was highly enriched with nuclei (panel a for

DNA-BINDING PROTEIN IN FISSION YEAST 89 1

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600 ( 6 3 ) 120

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S R I V Y S E I Q T K F G P F P F S T R U I S F D S R T N U G L N E C T S H K L ~ 3 0 3 ) ~ A ~ T - - A T X ; ~ T ~ ~ T A T ~ ~ A T T ~ T T ~ ~ ~ A C M T T A T ~ T C ~ A T T C ~ M C C T ~ G M ~ 1 4 4 0

L C P Y E V L L D K D G G I V A E F Y S T I A L T K K G T I I L S D S E P K E D ( 3 4 3 J m U t Z I M ~ T ~ ~ - ~ T - ~ C ~ - C ~ A ~ ~ G ~ ~ C M ~ - ~ C A ~ - T M T ~ T M f f i 1560

C I I S D K K V E D P E I V A L L L T P I K V T $ ~ K K ~ ~ K ~ P S K A N E ~ (381)

~ ~ T - ~ A r M T ~ ~ ~ ~ ~ ~ ~ G T T ~ ~ A T M ~ T ~ ~ ~ ~ M ~ C T T ~ A C T A T T C M C ~ A T T T ~ 1 680 Z T M ~ T ~ A ~ T ~ ~ ~ M G n T ~ T A T T ~ T T ~ ~ ~ G ~ A ~ M C A ~ T T T ~ M 1800 K T ~ ~ A ~ ~ T - T M ~ ~ G T ~ M T C C M ~ ~ A ~ ~ T A T ~ ~ ~ A T T M T 1905

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Figure 8. Nucleotide sequence of the S. poombe genomic DNA region encompassing the gene encoding the 42 K-protein. The determined N-terminal amino acid sequence for a tryptic fragment of the 42 K-protein is underlined in the predicted amino acid sequence. A possible nuclear targeting amino acid sequence is double-underlined. This sequence data will appear in the EMBLIGenBanWDDBJ Nucleotide Sequence Data Libraries under accession number D14907.

phase-contrast micrograph, panel b for DAPI- stained fluorescence micrograph). This preparation was then subjected to Western blot analysis with polyclonal antiserum raised against the 42 K- protein (Figure 11B). A cross-reactive band with the same electrophoretic mobility as that of the 42 K-protein was detected in this particular nuclear fraction (lane 3). This result suggested that the 42 K-protein appears to be located mainly, if not exclusively, in the nucleus. In this respect, it is worth mentioning that a possible nuclear targeting sequence was found in the amino acid sequence of the 42 K-protein, at its C-terminal end (KNKKKSKK, see Figure 8 (Dingwall and Laskey, 1991).

The gene encoding the 42 K-protein may not be essential for cell growth

To assess the biological significance of the 42 K- protein in vivo, a disruption mutation of the coding region for the 42 K-protein in S. pombe was con- structed. The ura4+ gene was inserted into the SalI site, which is located in the initiation codon for the 42 K-protein ORF (see Figure 7). This construction was used to transform an S. pombe diploid strain (TP4-1D h'lTP4-5A h -) to uracil prototro-

phy. Transformants were analysed by Southern hybridization to verify that this construction had replaced one of the corresponding alleles (data not shown). Diploids carrying one disrupted allele were then induced to undergo meiosis, and the tetrads were dissected. The result of tetrad analysis suggested that the gene encoding the 42 K-protein appears to be non-essential for cell growth under standard growth conditions (data not shown).

Conclusion A DNA curvature appears to be an important

determinant of both the function and the architecture of a nucleoprotein complex concerned in a variety of biological processes (Travers, 1989; Hagerman, 1990). In this study, we purified an S. pombe protein, tentatively named the 42 K- protein, which associates preferentially with a synthetic curved DNA sequence in vitro, although it also binds to non-curved DNA sequence with lower affinity. In other words, if the 42 K-protein can choose between the two types of DNA sequences, it preferentially associates with the curved one. Based on the results obtained by a series of DNA-binding assays (Figures a), we believe that the major determinant of the observed preferential


Figure 9. Expression of the 42 K-protein in E. coli. Strain JM83 was transformed either with pUSI-2 (control) or pOP42 (see Figure 7). The cells were grown in the presence of isopropyl P-D-thiogalactopyranoside (2 mM), and then harvested at the logarithmic growth phase. (A) Total cell lysates were prepared from these cells, and analysed by SDS-PAGE, followed by staining: lane 1, total cell lysate (pUSI-2); lane 2, total cell lysate (pOP42); lane 3, purified 42 K-protein. (B) The gel of SDS-PAGE, prepared under the same conditions as those for (A), was subjected to Western blot analysis with polyclonal mouse antiserum raised against the 42 K-protein.

S. pombe: 7 1 KDAYKGIAFPTAVSPNDMAAHLSPLKSDPEANLALKSGDVVKILLGAHIDG **** ** * * **** ** * * * * * * * * * * * * C. e l egans : 1 FWFTKGIAMPTCISIDNCICHYTPLKS--EAPWLKNGQWKVDLGTHIDG

S. pombe: 122 FASLVATTTWS---EEPVTGPAADVIAAASAALKAAQRTIKPGNTNWQVT * * ** *** ** ** * * * * * C. e legans: 50 LIATAAHTVWGASKDNKVTGKLADLLRGTHDALEIAIRSLRPDTENTTIT

S. pombe: 170 DIVDKIATSYGCKPVAGMLSHQQEREVIDGKKQVILNPSD

C. e legans: lOl KNIDKTkAEFGLTPIENMLSHQLERNEIDGEKEIIQNFWR ** * * * ***** ** *** * * *

Figure 10. The amino acid sequence alignment of the 42 K-protein of S. pombe and a hypothetical protein of C. eleguns. Note that only the N-terminal amino acid sequence of this C. eleguns protein is available (Waterston et al., 1992). Amino acid identity is indicated with asterisks.


Figure 1 1. Microscopic observation and Western blot analysis. (A) Microscopic observation of the isolated nuclear fraction: a, phase-contrast micrograph; b, fluorescence micrograph of the DAPI-stained nuclear fraction (see Materials and Meth- ods). (B) Western blot anlaysis. The purAed nuclear fraction (30 pg of protein) and the purified 42 K-protein (2 pg) were analysed on SDS-PAGE, followed by staining (lanes 1 and 2, respectively). These samples were also subjected to Western blot analysis with mouse antiserum raised against the 42 K- protein (lanes 3 and 4 respectively).

binding is an overall DNA curvature, rather than a specific nucleotide sequence (e.g. dAn stretches

and their flanking sequences). This binding of the 42 K-protein is observed at a relatively high protein to DNA ratio. This may be solely a matter of the in vitro DNA-binding activity of the purified 42 K-protein used in this study. Alternatively, it may suggest that the 42K-protein binds to the curved DNA by forming a polymeric nucleoprotein complex. However, at present it is difficult to estimate how many protein molecules bind per DNA molecule. The mode of interaction between the 42 K-protein and a cognate DNA sequence must await further clarification. In any event, the 42 K-protein, characterized in this study, seems to be a novel type of S. pombe DNA-binding protein.

The biological function of the 42 K-protein is not known at present. It was revealed that the 42 K-protein is located in the nucleus. The preliminary results, obtained with immunofluorescence microscopic analysis with antiserum against the 42 K-protein, suggested that this protein is located in the peripheral area of the nucleus (e.g. the nuclear membrane) (Yamada, Ueguchi and Mizuno, unpublished data). Therefore, it is tempt- ing to speculate that this curved DNA-binding protein may play a role in certain biological processes, such as DNA replication or DNA segre- gation during the meiotic or mitotic events that occur during proliferation of S. pombe. In this connection, it would be worth mentioning that the isolated functional centromeric DNA of S. pombe (namely, cenl) contains a strikingly curved DNA structure, i.e. a relatively large central portion of cenl, covering a 2.2-kb region, displays remark- able DNA curvature (Ueki et QZ., 1993). In any case, curved DNA sequences may provide preferential binding sites for the 42 K-protein, at which this protein may play a biological role. Thus, it is crucial to search for the naturally occurring target DNA in the S. pombe genomic DNA. Experiments along these lines are also currently underway in our laboratory.

ACKNOWLEDGEMENTS We wish to thank Drs M. Yanagida and 0. Niwa (Department of Biophysics, Kyoto University, Japan) for providing the genomic and cDNA libraries for S. pombe as well as a set of S. pombe strains, and helpful discussions. This study was supported by a Grant-in-Aid for Scientific Research on a priority area from the Ministry of Education, Science and Culture of Japan, and a special fund from the Taiko Foundation.


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A fission yeast gene encoding a protein that preferentially associates with curved DNA