Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press
The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast
Ken-ichi Mizuno/'' Yukihiro Emura/'^* Michel Baur,^ Jiirg Kohli,^ Kunihiro Ohta/'^ and Takehiko Shibata^ ^Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan; ^Applied Biology, Nihon University, Fujisawa, Japan; ^Institute of General Microbiology, University of Bern, Bern, Switzerland
The G ^ T transversion mutation, ade6-M26, creates the heptanucleotide sequence ATGACTG, which lies close to the 5' end of the open reading frame of the ade6 gene in Schizosaccharomyces pombe. The mutation generates a meiosis-specific recombination hot spot and a binding site for the Mtsl/Mts2 protein. We examined the chromatin structure at the ade6 locus in the M26 strain and compared it to that of the wild-type and hot spot-negative control M375. Micrococcal nuclease (MNase) digestion and indirect end-labeling methods were applied. In the M26 strain, we detected a new MNase-hypersensitive site at the position of the M26 mutation and no longer observed the phasing of nucleosomes seen in the wild-type and the M375 strains. Quantitative comparison of MNase sensitivity of the chromatin in premeiotic and meiotic cultures revealed a small meiotic induction of MNase hypersensitivity in the ade6 promoter region of the wild-type and M375 strains. The meiotic induction of MNase hypersensitivity was enhanced significantly in the ade6 promoter region of the M26 strain and also occurred at the M26 mutation site. The formation of the MNase-sensitive region around the heptamer sequence was abolished by the introduction of single-nucleotide substitutions in the heptamer sequence, which also abolish hot spot activity and binding of Mtsl/Mts2. These data suggest that Mtsl/Mts2 binding to the heptamer sequence results in a chromatin structure suitable for the recruitment of a meiosis-specific recombination function or functions. [Key Words: recombination; meiosis; yeast; hot spot; chromatin] Received December 20, 1996; revised version accepted February 18, 1997.
It has been demonstrated that meiotic recombination oc coccal nuclease (MNase) becomes more specific and curs preferentially at defined sites called hot spots on quantitatively more important at hot spots during early meiotic chromosomes in various eukaryotic organisms meiotic prophase (Ohta et al. 1994). This implies the (Shiroishi et al. 1993; Smith 1994; Lichten and Goldman action of meiosis-specific functions for chromatin 1995). In the budding yeast Sacchaiomyces ceievisiae, changes at hot spots. However, the existence of meiotic most of the meiotic recombination is initiated by the DSBs and the role of chromatin structure at hot spots formation of meiosis-specific DNA-double strand breaks have not been established in organisms other than S. (DSBs) at hot spots (Nicolas et al. 1989; Sun et al. 1989; ceievisiae. Cao et al. 1990; Zenvirth et al. 1992; de Massy and Nico In the fission yeast Schizosaccharomyces pombe, the las 1993; Goyon and Lichten 1993; Nag and Petes 1993; meiotic recombination hot spot ade6-M26 (M26) (Gutz Wu and Lichten 1994). Meiotic DSBs on chromosomes 1971) is created by a G-> T transversion in the open are preferentially introduced at nucleosome-free regions reading frame close to the 5' region of the ade6 gene that show hypersensitivity to nucleases (Ohta et al. (Ponticelh et al. 1988; Szankasi et al. 1988). The muta 1994; Wu and Lichten 1994; Fan and Petes 1996). There tion creates a nonsense codon and causes up to a 15-fold, fore, accessibility of DNA is a primary requirement for meiosis-specific elevation of recombination frequency hot spot activity. In addition, hypersensitivity to micro- compared to the ade6~M375 mutation (Gutz 1971; Pon ticelh et al. 1988; Schuchert and Kohh 1988). The mu tation ade6-M375 (M375) is a G ^ T base substitution that creates a nonsense mutation in the codon preceding *K. Mizuno and Y. Emura contributed equally to this work. ^Corresponding author. M26 (Ponticelh et al. 1988; Szankasi et al. 1988), but it E-MAIL [email protected]; FAX 81-48-462-4671. does not show hot spot activity (Gutz 1971), thus pro-
876 GENES & DEVELOPMENT 11:876-886 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press
chromatin remodeling in ade6-M26 hot spot viding an excellent negative control for M26. M26 has medium. The isolated chromatin was digested with vari features similar to hot spots in S. cerevisiae. (1) The ous concentrations of MNase. DNA was purified and M26-carrying chromatid is a preferential recipient of ge examined by agarose gel electrophoresis, followed by netic information: It is preferentially converted to wild- ethidium bromide staining. A ladder of several broad type in one-factor crosses (Gutz 1971). This is a typical bands indicated integrity of nucleosomal repeats in the and predicted feature of the DSB repair model (Szostak et chromatin fraction (data not shown). The MNase-di- al. 1983). (2) The conversion rates of markers upstream of gested DNA was then cleaved by appropriate restriction M26 increase steadily as their distance from M26 endonucleases, and the DNA fragments were analyzed decreases. This is concluded from the combination of by Southern hybridization using short probes for se tetrad data (Gutz 1971) with the physical analysis of con quences adjacent to one of the restriction sites. version tracts (Grimm et al. 1994), suggesting the exis Figure 1 illustrates the MNase-sensitive sites in chro tence of an initiation site in the 5' region of the ade6- matin of the ade6 locus in wild-type, M375, and M26 M26 gene. (3) The activity of M26 is dependent on the mutations at t = 3 hr after transfer of the cells into a chromosomal context. Transplacement of the whole sporulation medium. The MNase-digestion patterns of ade6-M26 gene to other sites in the genome often abol wild-type and M375 were similar. We observed two hy ishes the hot spot activity (Ponticelli and Smith 1992; persensitive sites (-210 and -80) in a presumed promoter Virgin et al. 1995), whereas the transplacement of the region that lies in the 5' intergenic region of the adeS ade6 gene with M26 sometimes leaves the hot spot ac locus and a ladder of sensitive sites in the open reading tive. In addition, M26 enhances recombination between frame of adeS. The regular spacing of average 144 bp plasmid versus chromosome, only when it is located on (seven nucleosomes at positions 90-1100) represents a the chromosome (Ponticelli and Smith 1992; Virgin et al. phasing of nucleosomes (Bernardi et al. 1991) as revealed 1995). These results imply a role for chromatin structure by comparison with the digestion patterns of naked or higher-order chromosome structure in the control of DNA. In contrast, the chromatin of M26 was signifi the M26 hotspot. cantly different. In addition to the two hypersensitive The M26 mutation generates the heptanucleotide se sites (-210 and -80) in the presumed promoter region, we quence (5'-ATGACGT-3', M26 mutation underlined). detected a novel hypersensitive site at the position of the All base substitutions at any of the seven positions in the M26 mutation (170) that is covered by a phased nucleo- heptamer abolish hot spot activity (Schuchert et al. some in wild-type and M375 mutations. Furthermore, 1991). The heterodimeric protein Mtsl/Mts2 was iden the regular spacing of -150 bp was not observed and tified as a factor binding specifically to DNA carrying the some new MNase-sensitive sites appeared at positions heptamer sequence (Wahls and Smith 1994). This bind corresponding to MNase-sensitive sites in the naked ing was abolished when single-base substitutions were DNA of the coding region (e.g., bands at 340, 480, and introduced into the heptamer sequences, showing that 670). there is perfect correlation between the sequence re quirements of the heptamer sequence for hot spot activ ity in vivo and Mtsl /Mts2-binding activity (Schuchert et M26-dependent enhancement of meiotic induction of al. 1991). Thus, the Mtsl/Mts2 protein could have a cru MNase hypersensitivity cial role in the establishment of the M26 hot spot. How Next, we compared the chromatin structure at the ade6 ever, the reason for the meiosis specificity of M26 still locus in premeiotic and meiotic cells (Figs. 2 and 3). To remains unexplained, as the activity of the Mtsl/Mts2 allow comparison, the radioactivity of each band was protein is present both in mitotic and meiotic cell ex quantified. The MNase sensitivity at each site was ex tracts (Wahls and Smith 1994). One possibility is that pressed relative to the sum of all band intensities in the meiotic alteration of chromatin structure may contrib lane. For M375, we found that MNase sensitivity at the ute to the establishment of the M26 hot spot. presumed promoter increased slightly during meiosis In this study we have examined the chromatin struc (2.4 ± 1.3-fold at site -210, 3.9 ± 2.2-fold at site -80, ture at the ade6 gene before and during meiosis of diploid n = 3; see Table 1). We observed the same features for the strains homozygous for M26, M375, or wild-type se wild type (Table 1). On the other hand, the sensitivity at quences. We found that the ade6 chromatin is remodeled other sites did not change significantly, for example, strikingly in M26 diploids but not in wild-type or M375 1.3 ± 0.4-fold {n = 3) and 1.2 ± 0.1-fold {n = 2) at site -80 diploids. in M375 and wild type, respectively. This phenomenon is similar to the observations made at hot spots for mei otic recombination in budding yeast (Ohta et al. 1994). Results The meiotic induction of MNase sensitivity was en hanced by up to 9.3 ± 0.6-fold as a result of M26 at site M26-dependent remodeling of chromatin structure -80 in the presumed promoter region [n = 3, see Fig. 3; We examined the meiotic chromatin structure at the Table 1), whereas the sensitivity at site 80 was not en ade6 region by an indirect end-labeling method. Chro hanced at all (1.0 ± 0.1-fold, n = 3). MNase sensitivity at matin was isolated from diploid strains homozygous for the M26 mutation site also increased significantly dur either wild-type, M375, or M26 at various time points ing meiosis (5.4 ± 2.2-fold, n = 3; see Fig. 3 and Table 1). (t = 0, 2,3, 4, 6 hr) after transfer of cells into a sporulation The increase in MNase hypersensitivity was first de-
GENES & DEVELOPMENT 877 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press
Mizuno et al.
Z ^ ■o " B , Xhol 1-485 ► —-485 -»-220^! Eco RI ATG 26 1231- —1231 870-^ -^340^ 750-^ -*-480-^! 580-^ 430-^ --670^ 250-^ -75fr^ — 842 90-^ ATG -80-^
-«1010»-| -210-*-
-«1100»-| -310-*-
-410
a 1469 -785^ Xhol Stul
Figure 1. Comparison of chromatin structures at the ade6 locus in wild-type, M375, and M26 strains. The yeast strains were harvested at t = 3 hr after transfer of cells into the sporulation medium. Chromatin and naked genomic DNA (from the M26 strain) were isolated, treated with MNase (50 and 32 U/ml, respectively), and analyzed by indirect end-labeling, as described in Materials and Methods. DNA was digested with Xhol [A] and StuI-£coRI (5), and subjected to Southern hybridization with probes for the sequence adjacent to the Xhol site at +1469 bp from the ATG of the adeS coding region (probe B in A), or for the sequence adjacent to the Stul site at -785 bp (probe C in B]. An interpretation of the nucleosome positions is shown. (Shaded shapes) Nucleosomes; (horizontal arrows) MNase-sensitive sites; (open triangles) the M26 mutation site. Vertical open arrows indicate the position of the coding region for the ade6 locus. The numbers beside the horizontal arrows indicate the distance from the A of the ATG at the beginning of the ade6 coding sequence. Sequences recognized by the hybridization probes are indicated by vertical open rectangles. The DNA size markers are Xhol (1953 bp), Xhol-Hindlll (627 bp), and Xhol-BamHl (1443 bp) for A, and Stul-EcoRl (2015 bp), Stul-ffindlll (1626 bp), and Stul-BamHl (810 bp) for B. Note that the M375 and M26 sequences differ only by single-base substitutions from the wild-type adeS sequence.
Figure 2. Quantitative comparison of pre- meiotic and meiotic chromatin structures at the ade6 locus in the M375 strain. Chro matin [(0 hr) premeiotic; (3 hr) meiotic] of the M375 strain was digested with 0, 7, 50, and 225 U/ml of MNase and examined as described in Fig. 1. DNA was digested with Xhol and hybridized with probe B (see Fig. lA). The radioactivity of each band was quantified by a Fuji BAS2000 system and Bio Image analyzer as described in Materi als and Methods. Band intensities in lanes containing samples digested with 50 U/ml of MNase were expressed as percentage of the sum of all band intensities. For further explanations, see Fig. 1. Intensity of a faint band corresponding to the band at 0 in Fig. 1 was not quantified in this study, as the intensity was too low for accurate quanti fication. (Open bars) Data of t = 0 hr,- 0 5 10 15 20 25 (hatched bars) data of t = 3 hr. The numbers beside the horizontal arrows indicate the Band Intensity (% of the total, in SOU lanes) distance from the A of the ATG at the be ginning of the ade6 coding sequence.
878 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press
chromatin remodeling in ade6-M26 hot spot
4' r.<, > A-. i V" T." «^ A"^ ^ '^ ■^'' 0^^ ^ ^ '?>'' -^ r^ S^^ ^ii^ ^ Figure 3. Quantitative comparison of pre- B -V, meiotic and meiotic chromatin structures ^ at the ade6 locus in the M26 strain. Chro ^ matin isolated from the M26 strain was di gested with 7, 50, and 225 U/ml of MNase i and examined as described in Figs. 1 and 2. For further explanations see, Fig. 2. Open tected at t ~ 2-3 hr, and the sensitivity was still at a high GACGT should affect M26-dependent chromatin re level at £ = 6 hr (data not show^n). Under the same culture modeling. To test this idea, we analyzed four strains: two conditions, premeiotic DNA synthesis occurred at t ~ 2- hot spot-active strains with a single-base substitution 6 hr as revealed by laser scanning cytometry (Fig. 4) and outside the heptanucleotide (7T and 16C; see Fig. 5), and flow cytometry (data not shown). We also verified that two hot spot-inactive strains with a single-base substi the completion of meiosis I occurred around t = 8 hr by tution within the heptanucleotide (13G and 14C; see Fig. staining nuclei with 4',6-diamidino-2-phenylindole 5) (Schuchert et al. 1991). The Mtsl/Mts2 protein binds (DAPI) (data not shown). These results suggest that the to double-stranded DNA (dsDNA) probes having either changes of chromatin structure occur at the premeiotic S of the substitutions 7T or 16C but not to those with phase. either 13G or 14C in a gel mobility retardation assay (Wahls and Smith 1994). In the hot spot-positive controls 7T and 16C, we de Integrity of the heptamei sequence is necessary for tected MNase-hypersensitive sites at the presumed pro M26-dependent chromatin remodehng moter and around the heptamer (Figs. 5 and 6). The en The appearance of a nuclease-sensitive region around the hancement of the meiotic induction of MNase hypersen M26 mutation site suggests that the binding of Mtsl/ sitivity in the 7T and 16C strains was also detected at Mts2 to the heptamer sequence may contribute to the site -80, and the sensitivity increased at the heptamer enhanced DNA accessibility around the heptamer. If this region (Table 1). On the other hand, the chromatin struc is the case, modification of the heptamer sequence AT- tures of the ade6 region in I3G or 14C DNA were dif- Table 1. Quantification of MNase hypersensitivity Strains Site -210 Site Site M26 Wild type 2.910.3(2.7,3.1) 4.1 ±2.4 (2.4, 5.8) M375 2.4+1.3(2.2,3.7, 1.2) 3.9±2.2(2.1, 6.3, 3.2) M26 2.6 + 0.7(3.1,3.0, 1.8) 9.3±0.6(8.7, 9.5, 9.8) 5.4 + 2.2(7.9,3.5,4. 7T 2.6 + 1.3(3.6, 1.7) 10.2 ±2.0 (11.6, 8.8) 4.7 ±0.5 (5.1, 4.4) 13G 1.9 ±0.4 (2.1, 1.6) 4.1 ±2.0 (5.5, 2.7) 14C 1.9 + 0.4(2.2, 1.6) 3.0 ±1.7 (1.8, 4.2) I6C 2.4 ±0.2 (2.5, 2.3) 11.5 ±1.6 (12.6, 10.4) 3.6 ±0.2 (3.7, 3.4) The MNase hypersensitivity in each strain was quantified by a Fuji BAS2000 system and a Bio Image analyzer as described in Materials and Methods and Figs. 2 and 3. Meiotic induction of MNase hypersensitivity is expressed as the ratio of the meiotic hypersensitivity to the premeiotic hypersensitivity at each site including standard deviations. Numbers after ± represent standard deviations of the measurement. Numbers in parenthesis show the data for each experiment. The experiments were repeated three times with the M26 and M375 strains and twice with the wild-type, the 7T, 13G, 14C, and 16C strains. The ratio of the MNase hypersensitivity is basically independent from the concentration of MNase up to 50 U/ml. GENES & DEVELOPMENT 879 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mizuno et al. 2C4C 2C4C 2C4C 199 624 § -e^eg 159 o B 119 .? ^17 IkittpU 50 100 150 50 100 150 50 100 150 50 100 150 PI fluorescence value PI fluorescence value PI fluorescence value PI fluorescence value Figure 4. Changes in cell DNA content during meiosis. DNA content per cell was monitored during the meiosis of diploids at 0, 2, 4, and 6 hr after transfer of cells into sporulation medium by quantifying fluorescence of nuclei stained with propidium iodide (PI) using an Olympus LSClOl laser scanning cytometer. The vertical axis represents the number of cells; the horizontal axis indicates the PI fluorescence value per cell. The positions for 2C and 4C DNA content are indicated. The majority of fission yeast cells are usually in the G2 phase under vegetative growth conditions (Bahler et al. 1993). Therefore, the DNA content at t = 0 hr was 4C. After the cells are shifted into meiosis, they traverse M phase and enter Gj phase around t = 1-2 hr and then undergo premeiotic DNA synthesis (t = 2-6 hr). FACS analysis gave identical results (data not shown). From the staining nuclei with DAPI, completion of meiosis I was estimated to occur around t = 8 hr, and elongated or horse-tail nuclei were observed at t ~ 2-8hr (data not shown). ferent from those in 7T or 16C. As expected, they were sults seem to be inconsistent with the data of Bernardi almost identical with those of the wild type and M375 and coworkers (1991), who found the chromatin in M26 cells; that is, the MNase-hypersensitive site at the hep- haploid vegetative cells to be very similar to that in wild- tamer was absent and meiotic induction of MNase hy type cells. We have confirmed their results by showing persensitivity at site -80 was reduced. The difference in that the nucleosome positioning in the ade6 region in the band patterns in these strains is not attributable to the haploid M26 strain during vegetative growth was the difference in MNase-sensitive sites on naked DNA, very similar to the patterns seen in diploid wild-type and as the heptamer mutations did not affect the band pat M375 strains (K. Mizuno, J. Kohli, and K. Ohta, unpubl.). terns on naked DNA (data not shown). Therefore, there Therefore, the remodeling of chromatin structure found is positive correlation between hot spot activity, binding in the mitotic cells (i.e., premeiotic cells at t = 0 in the of Mtsl/Mts2 to the heptamer, formation of an acces meiotic time course in this study) seems to be related sible DNA region around the heptamer, and enhanced closely to ploidy or some other factors. We are currently meiotic induction of MNase sensitivity. Interestingly, analyzing this phenomenon. the 16C construct yielded a novel pattern; that is, the We found that chromatin remodeling by M26 requires three positioned nucleosomes present in wild type and the same integrity of the heptamer sequence as that re absent in M26 reappeared in 16C but were displaced in quired for the binding of the Mtsl/Mts2 protein (Wahls the 5' direction. The novel phasing of these three nucleo and Smith 1994). Altered chromatin structure as a con somes seems to be directed by the MNase-hypersensitive sequence of sequence-specific DNA-binding proteins has site at M26 and the additional base substitution I6C been reported for transcription factors. The a2 repressor (Figs. 5 and 6). in 5. cerevisiae was shown to affect the positioning of nucleosomes at the promoter through binding to its tar get DNA sequence in vivo (Shimizu et al. 1991). The Discussion GAGA factor in Drosophila was shown to be involved in formation of nuclease hypersensitive sites and remodel Chromatin remodeling by the heptamer sequence and ing of chromatin at the heat shock-responsive element in the Mtsl/Mts2 protein vitro (Tsukiyama et al. 1994). Probably, the binding of The results presented here indicate that the heptamer Mtsl/Mts2 to the heptamer influences the binding of sequence ATGACGT affects nucleosome positioning, nucleosomes to this region, thereby altering nucleosome possibly by facilitating the binding of the Mtsl/Mts2 positioning (Fig. 7). In this respect, it would be interest heterodimeric protein. In M26 chromatin, the phasing of ing to analyze chromatin structure in M26 strains dis nucleosomes was affected around the heptamer and rupted for the genes coding for Mtsl and Mts2. downstream in the open reading frame of the ade6 gene. It is intriguing that a pattern of nucleosome phasing The MNase-sensitive sites characteristic of naked DNA downstream of M26 is restored, when the single-base were observed in the chromatin digestion, and a novel substitution 16C is added to the heptanucleotide. The MNase-hypersensitive site also appeared at the hep resulting chromatin structure, however, differs from the tamer. All of these changes in DNA accessibility were wild type in that the nucleosomes are shifted toward the the consequence of the single G ^ T base substitution 5' end. We propose that the 16C sequence forms a bound that created M26 from the wild-type sequence. Our re ary for nucleosome positioning. Although the mecha- 880 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Chromatin remodeling in ade6-M26 hot spot 5'- T G} GG llA TT GG AA" CC G TI] G A -3' should be noted that the 16C mutation also enhances the A binding of Mtsl/Mts2 (Wahls and Smith 1994) and in creases meiotic recombination frequency beyond the T G C C M26 level (Schuchert et al. 1991). B 7T 13G 14C 16C Meiotic induction and M26 enhancement of MNase hypersensitivity: imphcation for the meiosis specificity ■M26 of the M26 hot spot and a general model for the initiation of meiotic recombination at ade6 The mechanism that creates the meiosis specificity of the M26 recombination hot spot (Ponticelli et al. 1988; -M375 Schuchert and Kohli 1988) is not yet fully understood. The Mtsl/Mts2 protein and its heptamer-binding activ 7T 13G 14C 16C strains ity are present in extracts of both mitotic and meiotic J3 .S b- r cells (Wahls and Smith 1994). In addition, the MNase- '^ induction sensitive site at M26 is also detected in mitotic and mei inincn (Sf^SoS^oSr^o^o S< s MNase(Units) otic diploid cells, suggesting the constitutive interaction of Mtsl/Mts2 with the heptamer in vivo and the neces sity to invoke the existence of additional factors to ex M26sitet>l <|M26site plain M26 meiosis specificity. We have demonstrated here that MNase hypersensi tivity increases significantly at the presumed promoter and the heptamer sequence during early meiosis (Table I). The increase in MNase sensitivity was observed from two aspects: (1) Meiotic induction of MNase hypersen sitivity is observed in all strains (wild type, M375, and M26) at the two sites, -210 and -80, in the presumed promoter; and (2) in M26, meiotic induction of MNase hypersensitivity is significantly stronger at the -80 pro MNase hyper moter site and at the M26 site when compared to those sensitive site in wild-type and M375 strains. These are the first obser around the M26 site vations suggesting the interaction of meiosis-specific Figure 5. Integrity of the heptamer sequence is required for functions with accessible DNA regions during S. pombe M26-dependent chromatin remodeling. [A] The DNA sequence meiosis. We put forward three explanations for the meio- around the M26 heptamer (boxed) is shown together with the sis-specific functions that cause the meiotic increase in additional single-base substitutions (arrows) and the mutation the MNase sensitivity of the M26 hot spot. designations. [B] The correlation is shown between hot spot activity in vivo (Schuchert et al. 1991) and the DNA-binding The first possibility is that enhanced binding of Mts 1 / activity of Mtsl/Mts2 to each altered sequence in vitro (Wahls Mts2 to the heptamer during meiosis increases DNA and Smith 1994). The values for the in vivo data are normalized accessibility at M26. This is supported by the observa by setting the M26 recombination frequency as 100% and the tion that the MNase hypersensitivity at the heptamer M375 frequency as 0%. (C) The chromatin structures of the increased about fivefold during meiosis. Although this heptamer mutants were analyzed by indirect end-labeling as possibility is likely, it should be noted that the MNase- described in Fig. 1. The strain names {top line), the times of cell hypersensitive site at M26 is already present in mitotic harvest [middle line, (0 hr) premeiotic; (3 hr) meiotic], and the cells, in which M26 enhanced homologous recombina conditions for MNase digestion {bottom line, U/ml) are indi tion could not be detected at all (Ponticelli et al. 1988; cated. The position of the M26 mutation is marked by open Schuchert and Kohli 1988) unless a DSB is supplied at triangles. The presence (+) or absence (-) of the MNase-hyper- sensitive site at the M26 mutation site is shown below the gel. the ade6 region by introduction of HO endonuclease (Os- DNA size markers were as in Fig. lA. The difference of the band man et al. 1996). This observation implies that meiotic patterns in these strains is not attributable to the difference in activation of the M26 hot spot might include other steps MNase-sensitive sites on naked DNA, as the heptamer muta such as recruitment of a meiosis-specific nuclease or a tions did not affect the band patterns on naked DNA (data not recombination protein to accessible DNA regions cre shown). ated by Mtsl/Mts2 (see below). The second possibility is that changes in DNA structure occur at the heptamer and in the presumed promoter re gion during meiosis (e.g., melting of DNA yielding single- nism underlying this boundary formation is unclear, the stranded regions). However, in the case of budding yeast, 16C in the context of the local DNA sequence may we could not detect a significant change in DNA structure change the intrinsic DNA structure or recruit another during meiosis at the nucleosome-free regions of the ARG4 DNA-binding protein to form the boundary (Fig. 6). It hot spot by genomic footprinting analysis using single GENES & DEVELOPMENT 8S1 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mizuno et al. heptamer M26 site nucleosomes Type A) (5'-AGGACGT-3') (-210) (-80) (90) (250) (430) (580) (750) (870) (1010) (1100) Type B) (7)Mts2 AAl^ (5'-ATGACGT-3') ^^ T TW I T I (340) (450)1 (590)1(670)1 | | | (-210) (-80) (80) (170) (250) (480) (630) (750) (870) (1010) (1100) Type C) (5'-ATGACGTC-3') (-210) (-80) (80) (170) (350) (520) (670X760) (870) (980X1090) Figure 6. A schematic diagram showing nucleosome positions in different ade6 strains. The positions of nucleosomes were estimated from the data on MNase-digested chromatin and digests of naked DNA in Fig. 1. The nucleosome patterns were classified into three types: Type A includes wild-type, M375, 13G, and 14C; type B includes M26 and 7T; and type C includes 16C. Vertical arrows indicate the positions of MNase-sensitive sites. Hatched horizontal arrows represent the ade6 coding region. The numbers in parenthesis give the distances from the first A of the initiation codon of the ade.6 coding sequence. The positions of the heptamer sequences are indicated by thin solid boxes; the nucleosomes are indicated by hatched circles; the Mtsl/Mts2 protein is shown as two kinked ovals. A shaded oval with a question mark in type C represents a putative third factor that may be involved in the boundary formation by the 16C sequence. stranded DNA (ssDNA)-specific probes such as KMn04, gions showing repression of MNase sensitivity during mung bean, and PI nucleases (K. Ohta, A. Nicolas, and T. meiosis prior to the formation of meiotic dsDNA breaks. Shibata, unpubl.). Thus, changes in DNA structure might The appearance of meiosis-specific footprints was not be too subtle to cause the increase in MNase sensitivity. detected in a subset of meiotic DSB-deficient mutants Assuming that the same mechanism for meiotic enhance such as mrell. Therefore, we speculate that the meiotic ment of MNase hypersensitivity is operative in both induction of MNase sensitivity at hot spots in budding yeasts, changes in DNA structure, at least formation of yeast reflects an essential reaction caused by the inter ssDNA, is unlikely to explain the meiotic increase in action of recombination proteins with DNA prior to the MNase sensitivity of the M26 hot spot. DSB formation. It is also possible that the meiotic en The third explanation is that the meiotic increase in hancement of MNase hypersensitivity at the ade6-M26 MNase sensitivity at M26 and then hot spot activity is locus may reflect the action of meiotic recombination caused by the recruitment of other proteins to the ade6 enzymes. If meiotic recombination mechanisms are con gene that, unlike Mtsl/Mts2, would be specific for mei served between the two yeast species, rad32'^, the fission otic recombination (Fig. 7). Such recombination proteins yeast homolog of the MREll gene (Tavassoli et al. 1995), w^ould be recruited to the accessible DNA regions may be involved in the meiotic enhancement of MNase formed by the binding of Mtsl/Mts2 protein and also hypersensitivity. The study of the chromatin structure probably by transcription factors bound to the promoter. in yeast cells with a disruption of the iad32* or other This model is based on our results showing that MNase recombination genes will be a test of this model. hypersensitivity at hot spots increases significantly dur ing meiosis in budding yeast (Ohta et al. 1994). In addi tion, we carried out genomic footprinting analysis of Is the ATGACGT sequence enhancing a preexisting chromatin at the ARG4, CYS3, and yCi?4Swhot spots at recombination initiation sitel the single-nucleotide resolution level (K. Ohta, A. Nico In budding yeast, hot spots often coincide with acces las, M. Furuse, A. Nabetari, H. Ogawa, and T. Shibata, sible promoter regions that show meiotic induction of unpubl.). Meiotic induction of MNase sensitivity often MNase hypersensitivity (Ohta et al. 1994). The presence occurs at sites adjacent to (within a few base pairs) re of meiotically enhanced chromatin alterations in the 882 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press chromatin remodeling in ade6-M26 hot spot /^ transcription spot activity is abolished when the ade6 presumed pro factors nucleosome ade6 ORF moter region is deleted (Zahn-Zabal et al. 1995). This finding seems to be compatible with the interpretation promoter that M26 enhances an initiation site localized within the presumed promoter region and that no novel initiation site [single-strand break (SSB) or DSB] is created at the B heptanucleotide. However, we cannot exclude the pos Mts1 ade6-M26 ORF sibility that the deletion might change chromatin struc ture around the M26 mutation sites to inactivate its hot spot activity. It will be interesting to determine whether M26 heptamer the MNase-hypersensitive site at M26 is still present in meiosis chromatin isolated from an M26 strain carrying a dele Mtsi tion of the presumed promoter region. If the site at M26 is still found in strains deleted for the presumed pro moter, then an examination of the meiotic enhancement of MNase sensitivity at the M26 site in these strains Increase in DNA IMelosls-speclflc Factor? should provide an important clue about the role of the accessibility M26 mutation in the hot spot activity. In Figure 7 we propose an alternative idea that both the presumed promoter and the heptamer regions serve as initiation sites for meiotic recombination at the M26 locus. Assuming that meiotic induction of MNase sen DNA break ? sitivity is an obligatory signal for initiation, as is the case fo.t in S. cerevisiae, the presence of the induction in MNase Figure 7. A model explaining the meiosis specificity of the hypersensitivity after meiotic induction at the presumed M26 hot spot. (A) Without the heptamer, phasing of nucleo- promoter sites and the M26 heptamer is consistent with somes (hatched circles) occurs over the ade6 open reading frame the notion that both sites are initiation (DSB or SSB) (horizontal arrow). The presumed promoter region (hatched sites. According to this model, the M26 heptamer would rectangle) is constitutively open and can act as a natural recom play the double role of an enhancer of the background bination initiation site. The hatched hexagon indicates putative initiation site in the presumed promoter and of a novel transcription factors bound to the presumed promoter. [B] In the presence of the heptamer, binding of the Mtsl/Mts2 protein to hot spot of initiation at the heptamer. If the heptanucleo the heptamer alters the local chromatin structure to form an tide is contributing to the enhancement of the initiation accessible DNA region. (C) The accessible DNA region (espe site in the presumed promoter, then position effects of cially at the presumed promoter and the heptamer site) becomes the heptanucleotide would be expected. This could be a preferential target for meiosis-specific factors required for ini examined by varying the position of the heptanucleotide tiation of recombination. The recruitment of the meiosis-spe with respect to the presumed promoter and analyzing for cific factors causes meiotic enhancement in DNA accessibility any changes in chromatin structure. In relation to this, it (MNase hypersensitivity) at the presumed promoter and the is worthwhile noting that the heptamer still functions as heptamer sites. (D) Events for the initiation of meiotic recom a hot spot when it is moved to downstream positions in bination (such as DNA breaks) occur close to the presumed the ade6 gene (G. Smith, pers. comm.). promoter and/or the heptamer sites. Materials and methods ade6 presumed promoter region is consistent with the DNA and strains interpretation that an initiation site for meiotic recom bination exists in the presumed promoter region of the The plasmid containing the ade6-M26 sequence (pade6-M26) is wild-type ade6 gene and the M26 mutant. Previously, a derivative of pASl (Szankasi et al. 1988). The plasmid pade6- two possibilities have been advanced to explain the role M26 was digested with Xhol-EcoRl or Xhol-Stul to form 240- of the M26 mutation in the hot spot activity. First, the and 300-bp fragments. The fragments were subcloned into the Xhol-EcoRl site (pASI-1) or the Xhol-EcoRY site (pASI-2) in M26 mutation, and the resulting heptanucleotide that pBluescript(KS+), respectively. Probes for indirect end-labeling serves as a binding site for Mtsl/Mts2, enhances initia were prepared from pASI-1 and pASI-2 by digesting the plasmids tion of recombination at a site in the presumed promoter with Xhol-EcoRl followed by the purification of DNA frag region of ade6, which is also used for the background ments in agarose gels. All strains are from the collection at the recombination levels seen in M375. Second, the intro University of Bem (Switzerland) as summarized in Table 2. duction of the M26 mutation creates a new initiation site for recombination (Grimm et al. 1994). These two possibilities will not be distinguished until the physical Enzymes and chemicals nature and the sites of initiation events in M26 and Restriction endonucleases were purchased from New England M375 strains are known. Biolabs and Takara Shuzo Co. Ltd. MNase and Ficoll 400 were It has been demonstrated recently that the M26 hot from Pharmacia. Zymolyase lOOT was from Seikagaku Kogyo GENES & DEVELOPMENT 883 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mizuno et al. Table 2. Genotypes of strains Diploid Haploid mat ade6 ural leu3 ELD201 ELHIOI h* + ural-61 + (wild-type) ELH102 h- + + leu3-155 ELD203 ELH103 h* ade6-M375 uial-61 + (M375) ELH104 h' ade6-M375 + leu3-155 ELD205 ELH105 h^ ade6-M26 uial-61 + (M26) ELH106 h' ade6-M26 + leu3-155 ELD207 ELH107 h^ ade6-M26/7T ural-61 + (7T) ELH108 h' ade6-M26/7T + leu3-155 ELD209 ELH109 h* ade6-M26/13G ural-61 + (13G) ELHllO h' ade6-M26/13G + leu3-155 ELD211 ELHlll h^ ade6-M26/14C ural-61 + (14C) ELH112 h- ade6-M26/14C + leu3-155 ELD213 ELH113 h* ade6-M26/16C ural-61 + (16C! ELH114 h' ade6-M26/16C + leu3-155 All strains are from the Bern collection. The construction of the heptamer mutants was described by Schuchert et al. (1991). Co. Ltd. Proteinase K in solution was from Boehringer Mann cells were incubated for 15 min at 30°C with gentle agitation heim, RNase A was from Sigma. and resulting spheroplasts were pelleted. All subsequent steps were done at 0-4°C. The spheroplasts were then washed once in 5 ml/gram of ice-cold 1 M Sorbitol and resuspended well by Cell culture and sporulation pipetting in 7 ml/gram of freshly prepared lysis buffer [18% Standard media, presporulation, and sporulation were as de Ficoll-400, 10 mM KH2PO4, 10 mM K2HPO4 at pH 6.8, 1 mM scribed (Bahler et al. 1991, 1993). Briefly, diploid colonies were MgCli, 0.25 mM EGTA, 0.25 mM EDTA, 1 mM phenylmethyl- selected on minimal medium plates containing phloxin B. Colo sulfonyl fluoride (PMSF)]. After centrifugation at 20,000g for 40 nies showing efficient and synchronous sporulation were fur min, the crude nuclear pellet was resuspended well in 8 ml/ ther selected. Cells were grown in 1 liter of PM (Watanabe et al. gram of buffer A (10 mM Tris-HCl at pH 8.0, 150 mM NaCl, 5 1988) containing supplements with vigorous aeration at 30°C to mM KCl, 1 mM EDTA, 1 mM PMSF). One-milliliter aliquots of a cell density of 0.8 x 10^ to 0.9 x 10^, washed once in PM-N the crude chromatin suspension were digested with different (Watanabe et al. 1988). Half of the cells were precipitated and amounts of MNase (0, 7, 10, 20, 30, 50, and 225 U/ml) for 5 min frozen in liquid nitrogen, and then stored at -85 °C. The result at 37°C in the presence of 5 mM CaClj. The reaction was ter ing half of the cells were incubated with vigorous aeration at minated by adding 20 mM EDTA, 1 % (wt/vol) SDS, and 20 vig of 30°C in 0.5 liter of PM-N containing supplements. About 0.6- proteinase K and incubated at 50°C for 2 hr. Insoluble material 1.0 gram (wet weight) of cells was recovered in the standard was removed by microcentrifugation at room temperature at condition. The progression of cells into meiosis was monitored 15,000 rpm for 10 min. The supernatants were extracted twice by counting the number of nuclei in cells and determining the with phenol/chloroform/isoamyl alcohol, digested with RNase percentage of deformed and elongated nuclei (horse-tail nuclei) A, and then extracted once with phenol/chloroform/isoamyl (Bahler et al. 1993; Chikashige et al. 1994) by DAPI staining and alcohol. The extracted DNA was precipitated by ethanol, rinsed fluorescence microscopy as described (Bahler et al. 1993). From in 70% ethanol, and resuspended in 100 pi of TE buffer (10 mM the staining of nuclei with DAPI (data not shown), completion Tris-HCl, 1 mM EDTA at pH 8.0). The typical yield of DNA of meiosis I was estimated to occur around t = 8 hr, and elon from 1 gram cells was -200 ]ig. gated or horse-tail nuclei were observed at t ~ 2-8 hr. Laser scan ning cytometry was performed on cells stained with propidium iodide by an Olympus LSClOl setup according to the manufac Digestion of naked DNA turer's instructions and Sasaki et al. (1996). Naked DNA samples were prepared as described above except that addition of MNase was omitted. Nine hundred microliters of buffer A was added to 100 pi of naked DNA (20-30 pg). The Preparation of chromatin and digestion of chromatin DNA diluted DNA was incubated with various amounts of MNase by MNase (5-60 U/ml) in the presence of 5 mM CaClj at 37°C for 5 min. The preparation of crude chromatin fractions from S. pombe The reaction was stopped as described above. The mixture was cells was done according to the method of Bemardi et al. (1991), treated with 5 pg of proteinase K at 50°C for 30 min, extracted with some modifications. For the quantitative comparison of with phenol/chloroform/isoamyl alcohol, and precipitated by MNase sensitivity, samples of chromatin were prepared from a ethanol. The final precipitate was resuspended in 100 pi of TE fixed amount of cells (0.6-1.0 gram wet weight) in each experi buffer. Optimal conditions for MNase digestion were deter ment. Briefly, 1 gram of cells was incubated in 2 ml of preincu mined by gel electrophoresis. bation solution (20 mM Tris-HCl at pH 8.0, 0.7 M 2-mercapto- ethanol, 3 mM EDTA) at 30°C for 10 min and washed once in 5 Indirect end-labeling ml of ice-cold 1 M sorbitol. The gram of cells was then precipi tated and resuspended in 5 ml of freshly prepared Zymolyase Indirect end-labeling was performed as described (Bernardi et al. solution (37.5 mM Tris-HCl at pH 7.5, 0.75 M sorbitol, 1.25% 1991; Ohta et al. 1994). Briefly, 10 pi of DNA (2-3 pg) was glucose, 0.5% (wt/vol) Zymolyase-lOOT, 6.25 mM EDTA). The digested completely by restriction endonucleases. The digested 884 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Chromatin remodeling in ade6-M26 hot spot DNA was ethanol-precipitated, separated by electrophoresis on Fan, Q.-Q. and T.D. Petes. 1996. Relationship between nucle- a 1.2% agarose gel (40 cm long) containing 0.5 pg/ml of ethid- ase-hypersensitive sites and meiotic recombination hot spot ium bromide in TAE buffer (0.04 M Tris/acetate, 1 mM EDTA at activity at the HIS4 locus of Saccharomyces ceievisiae. Mol. pH 8.0) at 80 V for 15-17 hr and alkali-transferred to nylon Cell. Biol. 16: 2037-2043. membranes (Hybond N+, Amersham) under vacuum using a Goyon, C. and M. Lichten. 1993. Timing of molecular events in Vacugene (LKB) apparatus. Membranes were prehybridized for 1 meiosis in Sacchaiomyces ceievisiae. Stable heteroduplex hr and hybridized for 24 hr according to Church and Gilbert DNA is formed late in meiotic prophase. Mol. Cell. Biol. (1984) with 0.5-1 ng/ml of labeled probes. Probes were labeled 13: 373-382. by the random priming method according to the manufacturer Grimm, C, J. Bahler, and J. Kohli. 1994. M26 recombinational (Pharmacia) using 3.7 MBq (100 ijCi) of [a-^^P]dCTP (sp. act. hotspot and physical conversion tract analysis in the ade6 3000 Ci/mmole) for 100 ng of DNA fragments. Radioactive gene of Schizosacchaiomyces pombe. Genetics 136: 41-51. DNA fragments were quantified using the imaging plates for Gutz, H. 1971. Site-specific induction of gene conversion in Fuji BAS2000 Image Analyzer combined with the whole-band Schizosacchaiomyces pombe. Genetics 69:317-337. program of Bio Image (Bio Image Co. Ltd). Band intensity was Lichten, M. and A.S.Fi. Goldman. 1995. Meiotic recombination expressed as a percentage of the total of all the band intensities hotspots. Annu. Rev. Genet. 29: 423-444. of a lane including the unbroken parental fragment. For com Nag, D.K. and T.D. Petes. 1993. Physical detection of heterodu- parison, the same number of bands in lanes for each sample was plexes during meiotic recombination in the yeast Saccharo quantified by the whole band program. The nucleotide positions myces ceievisiae. Mol. Cell. Biol. 13:2324-2331. of the MNase-sensitive sites were measured according to the Nicolas, A., D. Treco, N.P. Schultes, and J.W. Szostak. 1989. An auto-band detection algorithm of Bio Image. The nucleotide initiation site for meiotic gene conversion in the yeast Sac numbers were estimated from the mean values of at least four chaiomyces ceievisiae. Natuie 338: 35-39. independent measurements. Ohta, K., T. Shibata, and A. Nicolas. 1994. Changes in chroma tin structure at recombination initiation sites during yeast meiosis. EMBO J. 13: 5754-5763. Acknowledgments Osman, F., E.A. Fortunato, and S. Subramani. 1996. Double- strand break-induced mitotic intrachromosomal recombina We are grateful to Ms. Elisabeth Lehman for the construction of tion in the fission yeast Schizosacchaiomyces pombe. Ge the strains. We also thank Dr. Gerald Smith, Dr. Wayne P. netics 142: 341-357. Whals, and Dr. Jeff Virgin for sharing data in advance of publi Ponticelli, A.S. and G.R. Smith. 1992. Chromosomal context cation and stimulating discussions. We thank Yoshimasa Kiyo- dependence of a eukaryotic recombinational hot spot. Proc. matsu (Olympus Co. LTD) for the measurement of cell DNA Natl. Acad. Sci. 89: 227-231. contents by the laser scanning cytometer. This project was sup Ponticelli, A.S., E.P. Sena, and G.R. Smith. 1988. Genetic and ported by a grant from Human Frontier Science Program physical analysis of the M26 recombination hotspot of (RG493/95), a grant for the Biodesign Research Program from Schizosacchaiomyces pombe. Genetics 119:491-497. RIKEN, grants from the Ministry of Education, Science, &. Cul Sasaki, K., A. Kurose, Y. Miura, T. Sato, and E. Ikeda. 1996. ture, Japan, and grants from the Swiss National Science Foun DNA ploidy analysis by laser scanning cytometry (LSC) in dation and of the Sandoz Research Foundation. colorectal cancers and comparison with flow cytometry. Cy- The publication costs of this article were defrayed in part by tometiy 23: 106-109. payment of page charges. This article must therefore be hereby Schuchert, P. and J. Kohh. 1988. The ade6-M26 mutation of marked "advertisement" in accordance with 18 USC section Schizosacchaiomyces pombe increases the frequency of 1734 solely to indicate this fact. crossing over. Genetics 119: 507-515. Schuchert, P., M. Langsford, E. Kaeslin, and J. Kohli. 1991. A References specific DNA sequence is required for high frequency of re combination in the ade6 gene of fission yeast. EMBO J. Bahler, J., C. Schuchert, C. Grimm, and J. Kohli. 1991. Synchro 10:2157-2163. nized meiosis and recombination in fission yeast: Observa Shimizu, M., S.Y. Roth, C. Szent-Gyorgyi, and R.T. Simpson. tions with patl-114 diploid cells. Curr. Genet. 19: 445-451. 1991. Nucleosomes are positioned with base pair precision Bahler, J., T. Wzler, J. Loidl, and J. Kohh. 1993. Unusual nuclear adjacent to the a2 operator in Sacchaiomyces ceievisiae. structures in meiotic prophase of fission yeast: A cytological EMBO J. 10:3033-3041. analysis. /. Cell Biol. 121: 241-256. Shiroishi, T., T. Sagai, and K. Moriwaki. 1993. Fiotspots of mei Bernardi, F., T. Roller, and F. Thoma. 1991. The ade6 gene of the otic recombination in the mouse major histocompatibility fission yeast Schizosacchaiomyces pombe has the same complex. Genetica 88: 187-196. chromatin structure in the chromosome and in plasmids. Smith, G.R. 1994. Fiotspots of homologous recombination. Ex- Yeast 7: 547-558. peiientia 50:234-241. Cao, L., E. Alani, and N. Kleckner. 1990. A pathway for genera Sun, H., D. Treco, N.P. Schultes, and J.W. Szostak. 1989. tion and processing of double-strand breaks during meiotic Double-strand breaks at an initiation site for meiotic gene recombination in S. ceievisiae. Cell 61: 1089-1101. conversion. Natuie 338: 87-90. Chikashige, Y., D.Q. Ding, H. Funabiki, T. Haraguchi, S. Szankasi, P., W.D. Heyer, P. Schuchert, and J. Kohh. 1988. DNA Mashiko, M. Yanagida, and Y. Hiraoka. 1994. Telomere-led sequence analysis of the ade6 gene of Schizosacchaiomyces premeiotic chromosome movement in fission yeast. Science pombe: Wild-type and mutant alleles including the recom 264:270-273. bination hot spot allele ade6-M26. J. Mol. Biol. 204:917- Church, G.M. and W. Gilbert. 1984. Genomic sequencing. Proc. 925. Natl. Acad. Sci. 81: 1991-1995. Szostak, J.W., T.L. Orr-Weaver, R.J. Rothstein, and F.W. Stahl. de Massy, B. and A. Nicolas. 1993. The control in cis of the 1983. The double-strand-break repair model for recombina position and the amount of the ARG4 meiotic double-strand tion. Cell 33: 25-35. break of Saccharomyces ceievisiae. EMBO J. 12: 1459-1466. Tavassoli, M., M. Shayeghi, A. Nasim, and F.Z. Watts. 1995. GENES & DEVELOPMENT 885 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mizuno et al. Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: A gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 23: 383-388. Tsukiyama, T., P.B. Becker, and C. Wu. 1994. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature 367: 525- 532. Virgin, J.B., J. Metzger, and G.R. Smith. 1995. Active and inac tive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe. Genetics 141: 33-48. Wahls, W.P. and G.R. Smith. 1994. A heteromeric protein that binds to a meiotic homologous recombination hot spot: Cor relation of binding and hot spot activity. Genes & Dev. 8: 1693-1702. Watanabe, Y., Y. lino, K. Furuhata, C. Shimoda, and M. Yama- moto. 1988. The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 7: 761-767. Wu, T.-C. and M. Lichten. 1994. Meiosis-induced double-strand break sites determined by yeast chromatin structure. Sci ence 263: 515-518. Zahn-Zabal, M., E. Lehmann, and J. Kohli. 1995. Hot spots of recombination in fission yeast: Inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim. Genetics 140: 469-478. Zenvirth, D., T. Arbel, A. Sherman, M. Goldway, S. Klein, and G. Simchen. 1992. Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. EMBO }. 11: 3441-3447. 886 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast. K Mizuno, Y Emura, M Baur, et al. Genes Dev. 1997, 11: Access the most recent version at doi:10.1101/gad.11.7.876 References This article cites 35 articles, 16 of which can be accessed free at: http://genesdev.cshlp.org/content/11/7/876.full.html#ref-list-1 License Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top Service right corner of the article or click here. Copyright © Cold Spring Harbor Laboratory Press