Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes
2004; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7600184
ISSN1460-2075
AutoresKehkooi Kee, Reine U Protacio, Charanjit Arora, Scott Keeney,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle25 March 2004free access Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes Kehkooi Kee Kehkooi Kee Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Reine U Protacio Reine U Protacio Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Charanjit Arora Charanjit Arora Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Scott Keeney Corresponding Author Scott Keeney Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Kehkooi Kee Kehkooi Kee Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Reine U Protacio Reine U Protacio Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Charanjit Arora Charanjit Arora Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Scott Keeney Corresponding Author Scott Keeney Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA Search for more papers by this author Author Information Kehkooi Kee1, Reine U Protacio2, Charanjit Arora1 and Scott Keeney 1 1Molecular Biology Program, Memorial Sloan-Kettering Cancer Center and Weill Graduate School of Medical Sciences of Cornell University, New York, NY, USA 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA *Corresponding author. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, Box 97, 1275 York Avenue, New York, NY 10021, USA. Tel.: +1 212 639 5182; Fax: +1 212 717 3627; E-mail: [email protected] The EMBO Journal (2004)23:1815-1824https://doi.org/10.1038/sj.emboj.7600184 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins. Introduction Meiotic recombination proceeds through the formation and repair of DNA double-strand breaks (DSBs) and is coordinated with changes in higher order chromosome structures. This coordination is essential because reciprocal DNA exchanges are only one part of the physical connections between chromosomes (known as chiasmata) essential for accurate segregation in the first meiotic division: sister chromatid axes must also be locally separated and the axes of recombinant nonsister chromosomes must be reciprocally exchanged (Zickler and Kleckner, 1999; Blat et al, 2002). This ensemble of local DNA exchange and higher order chromosome structures works along with sister chromatid cohesion to ensure that homologous chromosomes remain associated until anaphase I. Meiotic chromosomes are organized into linear arrays of chromatin loops. The loop bases and associated proteins define a structural axis for each chromatid. Sister chromatid axes are closely joined and, at pachytene, are synapsed with the axes of the homologous sister pair, via the central element of the synaptonemal complex (SC). Cohesins bind preferentially to AT-rich sequences distributed regularly along the chromosome (Blat and Kleckner, 1999; Tanaka et al, 1999) and are components of the chromosome axis (Klein et al, 1999; Eijpe et al, 2000, 2003; Pelttari et al, 2001). These and other considerations lead to the conclusion that cohesin-associated DNA sequences define the bases of the loops. Meiotic DSBs occur preferentially in the sequences between cohesin binding sites, and are thus considered to be in the chromatin loops (Blat et al, 2002), but the recombinosomes that carry out recombination are intimately associated with the axes (Moens et al, 1998). One way to reconcile these facts is to propose that protein–DNA complexes at DSB sites in chromatin loops are recruited to the axis, either as a condition for DNA cleavage or after DSB formation (Zickler and Kleckner, 1999; van Heemst and Heyting, 2000). Thus, the axis/loop organization of meiotic chromosomes defines one level of spatial organization for DSB formation and repair. A second level of spatial organization is that chiasmata form preferentially in chromosome domains that correspond to GC-rich isochores, or ‘R-bands’ (Ashley, 1988; Holmquist, 1992; Eisenbarth et al, 2000; Kong et al, 2002). Much of this positional bias for recombination is dictated during DSB formation in yeast (Baudat and Nicolas, 1997; Gerton et al, 2000; Blat et al, 2002). This bias represents a large-scale position effect, because a recombination reporter placed at different positions takes on the properties of its location: insertions into cold regions give low DSB levels and insertions into hot regions give high DSB levels (Wu and Lichten, 1995; Borde et al, 1999). DSB formation is catalyzed by Spo11, a relative of archaeal topoisomerases. Spo11 is widely conserved, but it does not act alone: in Saccharomyces cerevisiae at least nine other gene products are also required for DSB formation (reviewed in Keeney, 2001). The list of DSB genes comprises a meiosis-specific set (SPO11 itself plus REC102, REC104, REC114, MEI4, and MER2); a group of genes also involved in DSB repair and other functions in vegetative cells (RAD50, MRE11, and XRS2); and a gene with roles in cytoplasmic RNA metabolism (SKI8/REC103). In addition, DSB formation is reduced in mutants lacking either of two structural components of meiotic chromosomes, Red1 and Hop1 (reviewed in Keeney, 2001). This requirement provides strong evidence that activity of the DSB machinery is coordinated with higher order chromosome structures. Because of the integration of recombination with other aspects of chromosome structure and dynamics, it is important to understand not only the interactions that connect the DSB proteins to one another, but also the temporal and spatial patterns that govern their association with meiotic chromosomes. This work focuses on Rec102 and Rec104. Neither has a known homolog in species aside from other ascomycetes, or sequence motifs that suggest a biochemical function. Genetic and immunoprecipitation studies connect Rec102 and Rec104 to each other and to Spo11 (Salem et al, 1999; Kee and Keeney, 2002; Jiao et al, 2003), suggesting that Rec102 and Rec104 directly promote DSB formation as part of a multiprotein complex with Spo11. Here we examine interactions between Rec102 and Rec104, and describe the distribution, timing, and genetic requirements for their binding to meiotic chromosomes. Results Physical and functional interactions between Rec102 and Rec104 Epitope-tagged versions of Rec102 and Rec104 were expressed under the control of their normal promoters. Rec102 tagged at the C-terminus with multiple copies of the myc or flag epitopes complemented a rec102 null mutant (Kee and Keeney, 2002). Tagging the C-terminus of Rec104 yielded a nonfunctional protein (data not shown), so the protein was tagged instead at its N-terminus; mycREC104 supported normal DSB formation, intragenic recombination, and spore viability (see Materials and methods). Rec104 protein expression was meiosis-specific, similar to Rec102 (Figure 1A). Steady-state Rec104 levels at 4 h in meiosis were reproducibly lower in rec102, ski8, and mre11 mutants and were elevated 1.5-fold in a mer2 mutant (Figure 1B). For Rec102 as well, ski8 and mre11 mutations gave a noticeable reduction in steady-state levels (Figure 1B). Because ski8 and mre11 mutations cause defects during vegetative growth, it is possible that they affect meiotic gene expression indirectly because of premeiotic defects. This caveat does not apply to the meiosis-specific rec102 mutation, however, so the fact that Rec104 required Rec102 to accumulate to normal levels underscores the close relationship between these proteins. Figure 1.Physical and functional interactions between Rec102 and Rec104. (A) Western blot analysis of a time course of mycRec104 expression. Data for Rec102myc are from Kee and Keeney (2002). (B) Steady-state levels of Rec102myc and mycRec104 in DSB-defective mutants. Whole-cell extracts at 4 h in meiosis were analyzed by Western blot. Blots were stripped and reprobed for Bdf1 as a loading control. (C) Rec104 phosphorylation. Denaturing whole-cell extract was prepared at 4 h in meiosis, then mycRec104 was immunoprecipitated and treated with lambda phosphatase alone or in the presence of inhibitors as indicated. Whole-cell extracts from wild-type (WT) and rec102Δ strains are included for comparison. (D) Yeast two-hybrid analysis of interactions between Rec102 and Rec104. lacZ reporter expression was assayed in strains carrying the indicated fusion constructs. Each value is the mean±s.d. of at least three determinations. (E) Co-immunoprecipitation of Rec102 and Rec104. Nondenaturing whole-cell extracts were prepared from REC102flag strains carrying either REC104 or mycREC104, then immunoprecipitated with anti-myc antibodies. Input extracts and antibody matrix eluates were analyzed by Western blotting with anti-myc and anti-flag antibodies as indicated. Download figure Download PowerPoint This relationship is further emphasized by the effect of a rec102 mutation on Rec104 phosphorylation. Rec104 from early meiotic cells (2 h) migrated as a doublet in SDS–PAGE, whereas only the slower-migrating form was observed in extracts from cells at 3 h onward (Figure 1A). The slower-migrating form was converted to the faster one by phosphatase treatment, indicating that Rec104 is a phosphoprotein (Figure 1C). Hypophosphorylated Rec104 remained abundant in a rec102 mutant (Figures 1B and C). Therefore, Rec102 supports normal Rec104 phosphorylation. Because the other DSB genes were not required, Rec104 phosphorylation does not depend on DSB formation. Rec102 and Rec104 interacted weakly but reproducibly in a yeast two-hybrid system (Figure 1D). Nondenaturing whole-cell extracts from meiotic cells expressing Rec102flag and either mycRec104 or untagged Rec104 were treated with DNase I to eliminate DNA and then immunoprecipitated with anti-myc antibodies. Rec102flag was enriched in the immunoprecipitate from the mycREC104 strain relative to the REC104 control strain (Figure 1E). Similar co-immunoprecipitation results were recently reported with different tagged versions of these proteins (Jiao et al, 2003). Therefore, Rec102 and Rec104 interact directly in vivo. Dynamics of Rec102 and Rec104 binding to chromosomes Rec102 is a nuclear protein that associates with chromatin during prophase (Kee and Keeney, 2002). To more precisely define Rec102 association with chromosomes, nuclear spreads were double-stained for Rec102myc and Zip1, a component of the SC central element (Sym et al, 1993). The Zip1 pattern revealed the extent of SC formation and thus the stage in meiosis of any given nucleus (Figure 2). Rec102 first appeared on chromosomes with a punctate or patchy appearance prior to any SC assembly, that is, at or before leptotene (Figure 2A). This result places Rec102 on the chromosomes at the time when DSBs are formed. The staining became brighter through zygotene as the protein accumulated in patchy regions across much of the chromatin (Figure 2B). Staining lasted into early pachytene, indicating that Rec102 persists on the chromatin well past the period when DSBs are made (Figure 2C). Figure 2.Localization of Rec102 to spread meiotic chromosomes. (A–D) Nuclear spreads from SKY212 (REC102myc) were stained with anti-Zip1 (red) and anti-myc (green) antibodies. (E–G) Spreads from SKY788 (REC102myc REC8HA) were stained with anti-HA (red) and anti-myc (green). Equivalent exposures of representative nuclei are presented, staged according to the extent of SC formation. Scale bars, 3 μm (A–G) or 0.75 μm (H–K). (A) Leptotene or preleptotene. (B) Zygotene (a blow-up of the indicated region is shown in (H)) (C) Early pachytene with extended SCs and significant Rec102 staining (blow-up view in (I)) (D) Late pachytene with more compact SCs and lack of detectable Rec102. (E) Leptotene. (F) Zygotene (blow-up view in (J)). (G) Late zygotene/early pachytene (blow-up view in (K)). Arrows, Rec102 staining overlapping axes; arrowheads, Rec102 staining separated from axes. (L) Overlap of Rec102 immunofluorescence signal with Rec8 in leptotene and early zygotene versus late zygotene and early pachytene spreads. Diamonds, values for individual spreads; squares, mean±s.d. (M) Estimate of random colocalization. The Rec102–Rec8 overlap was measured after 180° rotation of the Rec102 immunofluorescence channel. Download figure Download PowerPoint Under these spreading conditions, pachytene nuclei could be divided into two classes: those with longer, overlapping SCs (Figure 2C) and those with more compact, distinct SCs (Figure 2D). A total of 28 pachytene nuclei were examined in detail. All (14/14) of the nuclei with extended SCs retained at least some Rec102 staining, whereas all (14/14) of the nuclei with more compact SCs were negative for Rec102 staining (compare Figures 2C and D). Because nuclei of both classes often lay side by side, it is unlikely that the differences between them are an artifact of spreading or uneven staining. We interpret the two classes to represent distinct stages of pachytene, with the more compact class later. Rec102 thus dissociates from the chromosomes during a specific transition period between the two pachytene stages. Rec104 immunostaining patterns were very similar to those of Rec102 (Figure 3). The protein first showed faint, patchy staining at leptotene, accumulated to maximal levels at zygotene, and disappeared during pachytene. Figure 3.Localization of Rec104 to spread meiotic chromosomes. Nuclear spreads from SKY791 (mycREC104) were analyzed by indirect immunofluorescence with anti-Zip1 (red) and anti-myc (green) antibodies. Scale bar, 3 μm. (A) Leptotene or preleptotene. (B) Zygotene. (C) Early pachytene. (D) Late pachytene. Download figure Download PowerPoint Spatial organization of chromosomal Rec102 Rec102 signals showed only partial overlap with Zip1 in zygotene cells, suggesting that at least a fraction of the Rec102 protein was located in the chromatin loops rather than on the axes, at least in the regions where SC had formed (Figures 2B and H). To further examine Rec102 localization, we double-stained nuclei for Rec102myc and Rec8-HA, a meiosis-specific cohesin that is axis-associated prior to SC formation (Klein et al, 1999; Blat et al, 2002; Eijpe et al, 2003). Representative images are shown in Figures 2E–G. Again, the early Rec102 signal overlapped only partially with the chromosome axes (Figures 2E, F and J). The staining patterns of both Rec8 and Rec102 are often irregular rather than being limited to discrete foci. We therefore evaluated overlap between these proteins on a pixel-by-pixel basis using the fluorescence intensities for each protein as recorded by the CCD camera. Based on visual inspection of the images, thresholds were set to define individual pixels as positive or negative for Rec8, and separately as positive or negative for Rec102. Then, the total fluorescence signal over background for Rec102 was measured and divided into two categories: the portion falling within Rec8-positive pixels (i.e. overlapping with axial staining) and the portion falling within Rec8-negative pixels (i.e. non-overlapping). In control experiments, Zip1 showed ∼90% overlap with Rec8 by this method (data not shown). When this analysis was applied to cells in leptotene through early zygotene, 53.7±7.7% of the total Rec102 signal overlapped with Rec8 staining (mean±s.d., N=16; Figure 2L). Put another way, almost half of the total Rec102 is clearly not colocalized with Rec8. Thus, Rec102 is broadly distributed across large chromosome regions early in prophase, and a substantial fraction of it is in the chromatin loops. Because of limits on resolution of the fluorescence signal, colocalization tends to be overestimated when two proteins lie nearby but are not truly colocalized. Therefore, our data are likely a conservative estimate of the size of the non-axial Rec102 population. The molecular nature of the Rec102 subpopulation that overlaps with Rec8 staining is less clear. One trivial possibility is that fortuitous overlap results from confinement of two unrelated protein distributions within the area of a nuclear spread (Gasior et al, 1998). To test this possibility, we measured overlap in a set of symmetrically spread leptotene nuclei after rotating the Rec102 image 180°. The rotated spreads showed overlap of 32.7±6.5%, decreased from 51.1±4.0% for the true images of these nuclei (Figure 2M; statistically significant at P<0.0001, Student's t-test). Thus, although fortuitous overlap may account for a fraction of the Rec102–Rec8 colocalization, it cannot account for all of it. It is possible that a subpopulation of Rec102 is intimately associated with axes. Alternatively, Rec102 and Rec8 may be components of distinct structures (e.g. chromatin loops versus axes) that only appear to colocalize because they are spatially coordinated with one another by virtue of being parts of a larger entity. The small size of yeast chromosomes and limits on the resolution of light microscopy preclude distinguishing between these possibilities. For cells later in meiotic prophase (late zygotene through early pachytene), a substantial fraction of Rec102 was still spatially separated from axes (arrowheads in Figures 2I and K), but there was an increase in the overlapping fraction to 63.2±9.6% of total Rec102 (N=25; Figure 2L; arrows in Figures 2I and K). The difference between earlier and later cells is statistically significant at P<0.002. Analogous changes in spatial distribution have been reported for Spo11 and Ski8 in Sordaria (Storlazzi et al, 2003; Tessé et al, 2003). This difference may reflect a change in the steady-state distribution such that more Rec102 is axis-associated later in meiosis. However, we note that the chromatin tends to be more compact at these later stages (see DAPI images in Figures 2C and G), so apparent overlap of distinct but spatially correlated populations would be expected to increase under these circumstances. Genetic requirements for recruitment to meiotic chromatin We next tested whether any of the other DSB proteins were required for the association of Rec102 and Rec104 with chromatin, using a subcellular fractionation assay outlined schematically in Figure 4A. In wild-type cells, ∼70% of Rec102 was in the first pellet, consistent with nuclear localization (Figure 4A). Most of the protein remained insoluble after detergent extraction, but was solubilized by DNase I digestion (fraction S3), indicating that the majority of Rec102 is chromatin-associated. In spo11 and rec104 strains in contrast, ∼90% of the Rec102 protein was recovered in the first soluble extract and little if any was recovered in the chromatin fraction (S3) (Figure 4B). The remaining ∼10% of the protein was recovered in the final pellet, and presumably represents nonspecific aggregation and/or unlysed cells. A similar pattern was observed in a ski8 mutant, except that a small portion (10–15% of total Rec102) was consistently recovered in the chromatin fraction. Thus, Rec102 chromatin association requires Rec104, Spo11, and, to a lesser extent, Ski8. A DSB-defective spo11 missense mutant (spo11-Y135F) supported nearly normal Rec102 fractionation behavior (Figure 4B), indicating that Spo11 protein is required, but not the ability to make DSBs. None of the other DSB proteins was required. Figure 4.Genetic requirements for chromatin association of Rec102 and Rec104. (A) The subcellular fractionation assay is outlined schematically at the top. The distribution of Rec102myc at 4 h in meiosis in a wild-type strain was followed by Western blotting with anti-myc antibodies. Lanes contain equal cell equivalents of each fraction. Tubulin illustrates the behavior of a soluble protein in this assay; the transcription factor Bdf1 demonstrates the behavior of a known chromatin-associated protein. (B) Effect of DSB-defective mutations on Rec102myc chromatin association. The indicated mutants were analyzed at 4 h in meiosis. (C) Behavior of mycRec104 in the subcellular fractionation assay in a wild-type background at 4 h in meiosis. Endogenous protease activities degrade mycRec104 to yield an ∼44 kDa species. (D) Effect of DSB-defective mutations on mycRec104 chromatin association. The first lysis step of the subcellular fractionation assay was carried out in mycREC104 strains carrying the indicated mutations. The yield of mycRec104 in the pellet fraction for each strain (as percent of total mycRec104) is given. Download figure Download PowerPoint Analysis of Rec104 proved more complicated because the protein was degraded during the assay (Figure 4C). Roughly half of the protein remained in the first pellet, consistent with nuclear localization of at least a fraction of the protein, and a prominent proteolytic product of 44 000 Mr could be solubilized by DNase I treatment, consistent with chromatin association. The protein was relatively stable until exposed to nonionic detergent (data not shown), perhaps reflecting activation of proteases. We therefore carried out only the first (detergent-free) lysis step to determine the effects of various mutants. Whereas ∼40–50% of total Rec104 protein partitioned to the pellet in wild-type cells and in mer2, mre11, rec114, and mei4 mutants under these conditions, yields in the pellet were reduced approximately two-fold in spo11 and ski8 mutants (Figure 4D). The pellet fraction was also reduced in a rec102 mutant, on top of the reduction in steady-state protein levels noted above. Whole-cell immunostaining extended these observations. In wild-type cells, Rec102 was predominantly nuclear prior to the first meiotic division as previously described (Kee and Keeney, 2002), but was dispersed throughout the nucleus and cytoplasm during prophase I in a rec104 mutant (Figure 5A). Nuclear localization was also impaired in spo11 and ski8 mutants, but to a different extent: 60–70% of cells showed diffuse staining throughout the cell but 30–40% showed discernible enrichment in the nucleus in addition to diffuse cytoplasmic staining (Figure 5A). Likewise, Rec104 protein was nuclear in wild type, dispersed throughout the cell in a rec102 mutant, and showed a mixture of staining classes for spo11 and ski8 mutants (Figure 5B). Thus, Rec102 and Rec104 are fully interdependent for nuclear localization and chromatin association and share at least a partial requirement for Spo11 and Ski8. These results further define functional subgroups among the DSB proteins, specifically, Rec102+Rec104 and Spo11+Ski8. Figure 5.Nuclear localization of Rec102 and Rec104. Cells from wild type and the indicated mutant strains carrying REC102myc (A) or mycREC104 (B) were fixed at 4 h in meiosis and stained with anti-myc antibodies (green) and with DAPI (shown in red). Representative cells are shown for the three staining classes observed: exclusively nuclear localization, diffuse staining of the entire cell, or diffuse cytoplasmic staining along with discernible enrichment in the nucleus. For each strain, ⩾50 cells were analyzed. Download figure Download PowerPoint Preferred binding sites for Rec102 are not restricted to recombinationally hot domains of chromosome III To examine Rec102 localization at higher resolution, we performed in vivo crosslinking and chromatin immunoprecipitation (ChIP). Immunoprecipitated DNA was 32P-labeled and hybridized to an array of 133 DNA fragments (average length ∼3 kb) spaced along chromosome III. This approach was previously used by others to study the localization of proteins important for DSB formation and repair and/or for higher order chromosome structure (Blat and Kleckner, 1999; Blat et al, 2002). To control for timing variations, cultures were staged relative to the first meiotic division (see Materials and methods). For cultures in which few cells had reached zygotene, Rec102 ChIP signals were only slightly over background (data not shown), so these samples were not analyzed further. Cultures with mostly leptotene/zygotene cells or later gave substantial Rec102 ChIP signals. One set of cultures analyzed (Figure 6A) contained cells half way through prophase I. Based on the timing of divisions in these cultures and the immunofluorescence patterns described above for independent cultures, we infer that the Rec102 ChIP signal was derived primarily from leptotene and zygotene cells. However, we cannot rule out the possibility that these cultures contain a significant subpopulation of early pachytene cells, which would also contribute to the observed patterns. In the second set of cultures (Figure 6B), half the cells had reached the first division. Because Rec102 disappears from chromosome spreads during pachytene, we infer that the ChIP signal in these later cultures was derived from the slower cells in the population that were still in late zygotene or early pachytene. Figure 6.ChIP analysis of the distribution of Rec102 on chromosome III. (A) Absolute signals for two cultures of a myc-tagged strain relative to cultures of an isogenic untagged strain. Each hybridization signal is plotted at the position of the probe fragment midpoint. The REC102myc cultures represent mostly leptotene and zygotene cells. (B) ChIP analysis of two independent REC102myc cultures representing mostly late zygotene and early pachytene cells. (C) Average of the two early REC102myc cultures (from panel A) compared to the average of the two later REC102myc cultures (from panel B). (D, E) Low-resolution views. In the cartoon of chromosome III at the bottom of the panel, the dark gray regions indicate the DSB-hot domains, the white regions are the DSB-cold domains, and the white circle indicates the centromere. (D) Relative abundances for Dmc1 and Red1 ChIP (from Blat et al, 2002) averaged in a nine-fragment sliding window (equivalent to ∼30 kb) and compared to sequence composition (%G+C content averaged over a 30 kb sliding window). (E) Similar sliding window analysis of the early Rec102 average (blue line) and the late Rec102 average (red line), compared to sequence composition. Download figure Download PowerPoint The Rec102 binding pattern showed a series of peaks and valleys across the length of chromosome III, well above background levels in control cultures lacking myc-tagged protein (Figures 6A and B). Peak positions were reproducible when cultures at the same stage were compared. However, early cultures differed significantly from late cultures, with only 35–45% peak overlap compared to 70–80% overlap for cultures of similar timing (Figure 6C; discussed further below). These results indicate that Rec102 binds preferentially to specific sites or regions in the genome, but that the array of preferred sites varies during the course of meiosis. The later cultures also showed higher overall Rec102 signals (Figure 6C). Early and late Rec102 binding patterns differed from previously established meiotic features of chromosome III, and did not meet the simplest expectation if the only role of Rec102 were to act locally to promote DSB formation. Previous studies defined the binding patterns for mitotic cohesins Smc1 and Mcd1/Scc1, for Red1 (a structural component of meiotic chromosomes), and for Dmc1 (a strand exchange protein required to repair meiotic DSBs) (Blat and Kleckner, 1999; Blat et al, 2002). At low resolution, assessed by averaging ChIP signals in a sliding window of nine probe fragments (∼30 kb), Red1 and Dmc1 bound preferentially within two large domains, one in each of the chromosome arms (Figure 6D). These recombinationally hot regions correspond to relatively GC-rich isochores (Figure 6D) (Baudat and Nicolas, 1997; Blat and Kleckner, 1999; Blat et al, 2002). In contrast, Rec102 was distributed more uniformly, with no obvious pre
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