Summary : The repair of DNA double-strand breaks (DSBs) requires the activity of the Mre11/Rad50/Xrs2(Nbs1) complex. In Saccharomyces cerevisiae, this complex is required for both the initiation of meiotic recombination by Spo11p-catalyzed programmed DSBs and for break end resection, which is necessary for repair by homologous recombination. We report that Mre11p transiently associates with the chromatin of Spo11-dependent DSB regions throughout the genome. Mutant analyses show that Mre11p binding requires the function of all genes required for DSB formation, with the exception of RAD50. However, Mre11p binding does not require DSB formation itself, since Mre11p transiently associates with DSB regions in the catalysis-negative mutant spo11-Y135F. Mre11p release from chromatin is blocked in mutants that accumulate unresected DSBs. We propose that Mre11p is a component of a pre-DSB complex that assembles on the DSB sites, thus ensuring a tight coupling between DSB formation by Spo11p and the processing of break ends.
Full text article at Molecular Cell.
Supplemental Figures (click on the pictures to enlarge) :
Figure S2: Genomic map of Spo11-dependant double-strand break (DSB) formation sites.
To examine the genome-wide localization of preferential DSB formation sites during meiosis, we used a com1/sae2 strain (ORD7311) in which meiotic DSBs remain unresected with Spo11p covalently bound to break ends [1-3] , allowing their detection by immunoprecipitation of the epitope-tagged Spo11-HA3His6 protein [4, 5] , without prior crosslinking, at t=6h after transfer into sporulation medium. The purified chromatin sample was compared to a whole cell extract (WCE) reference sample by hybridisation on a microarray containing PCR-amplified yeast coding regions as probes. For each chromosomal region represented by one ORF, we calculated the average enrichment ratio (ratio between the signal intensities of the precipitate and of the WCE reference) from two independent experiments after normalization and log-transformation.
ORFs have been organized along the chromosomes according to their relative position (median of the chromosomal coordinates). Centromeres are indicated by a black dot. Vertical bars report the enrichment ratio at each position, thus a high positive ratio indicates that DSB formation frequency is above average in the region of the corresponding ORF (DSB hot spots) and a negative ratio indicates that frequency is lower than average (cold spots). Given the average size of the hybridised DNA fragments and of the PCR probes spotted on the microarrays, the mapping resolution is of about 1kb.
Figure S1. Genomic map of Mre11p chromatin association sites.
We examined the genome-wide localization of Mre11p chromatin association sites, in meiotic com1/sae2 cells at t=6h, by immunoprecipitation using an anti-Mre11p antibody (J. Petrini) after a cross-linking treatment with formaldehyde. The purified chromatin sample was compared to a whole cell extract (WCE) reference sample by hybridisation on a microarray containing PCR-amplified yeast coding regions as probes. For each chromosomal region represented by one ORF, we calculated the average enrichment ratio (ratio between the signal intensities of the precipitate and of the WCE reference) from seven independent experiments after normalization and log-transformation.
ORFs have been organized along the chromosomes according to their relative position (median of the chromosomal coordinates). Centromeres are indicated by a black dot. Vertical bars report the enrichment ratio at each position, thus a high positive ratio indicates that Mre11p binding frequency is above average in the region of the corresponding ORF and a negative ratio indicates that frequency is lower than average. Given the average size of the hybridised DNA fragments and of the PCR probes spotted on the microarrays, the mapping resolution is of about 1kb.
References :
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Prinz, S., A. Amon, and F. Klein, Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae. Genetics, 1997. 146 (3): p. 781-95.
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Kee, K. and S. Keeney, Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae. Genetics, 2002. 160 (1): p. 111-22.
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Gerton, J.L., et al., Inaugural article: global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A, 2000. 97 (21): p. 11383-90.
Figure S3: Telomere and centromere repression of Mre11p and Spo11p chromatin association.
Average ratios of Mre11p or covalently-bound Spo11-HA3His6p binding were grouped in small distance intervals (1kb for telomeres and 0.5kb for centromeres) starting from a telomere or a centromere. If there were no influence of the centromere or telomere, one would expect to observe statistical fluctuation randomly distributed around zero. Linear regression model (blue line) has been used to fit intensities of protein binding in the selected regions around the centromere and telomere. The fitted values of the slope with the correspondent p value (the probability to obtain such a slope in the case when there is no preference in protein binding with respect to the positions of centromere and telomere) are indicated above each graph. We have also selected the regions (indicated by a vertical red line) around the centromere and telomere, where the overall ratio of binding is the most significantly lower than the overall intensity in the other part of the region. The amount of signal to the left of the red line is significantly smaller than to the right, as indicated by a negligible p value.
Supplemental Data :
Table S1. Mre11 hot spots. The average ratio from the seven experiments performed in the sae2 strain ORD7311 is indicated for each ORF segment. The rank corresponds to the median of ranks obtained in each experiment, as described in Experimental Procedures.
Download Table S1 (Excel sheet)
Table S2. Spo11 hot spots. The average ratio is from the two independent experiments performed in the sae2 strain ORD7311. Spo11 and Mre11 ranks were derived from the median of the ranks from each experiment, as described in Experimental Procedures. Previous Spo11 hot spots and their corresponding rank are from (Gerton, J. L., DeRisi, J., Shroff, R., Lichten, M., Brown, P. O., and Petes, T. D. (2000). Proc Natl Acad Sci U S A 97, 11383-11390).
Download Table S2 (Excel sheet)
Table S3. Comparison of the average ratios of Mre11 hot spots, at 6h and 0h in meiosis, with the strongest 300 Spo11 hot spots and previously published Spo11 hot spots. Ranks are as in Tables S1 ans S2. Previous Spo11 hot spots are from (Gerton, J. L., DeRisi, J., Shroff, R., Lichten, M., Brown, P. O., and Petes, T. D. (2000). Proc Natl Acad Sci U S A 97, 11383-11390).
Download Table S3 (Excel sheet)
Table S4. Strains used in this study. All strains are from the SK1 background, and are homozygous for ura3 lys2 ho::LYS2. Sources and/or references for mutant alleles: SPO11-HA3-His6::KanMX and rec102D::URA3 (Kee and Keeney, 2002); rad50D::hisG (Alani et al., 1989); rec104D::LEU2, rec114D::KanMX and mei4D::URA3, F. Klein; mre11DC49::URA3 (Furuse et al., 1998); mer2D::hisG-URA3-hisG, H. Ogawa; mre11-58S (Tsubouchi and Ogawa, 1998); ndt80D::LEU2, K. Benjamin; spo11D::hisG-URA3-hisG, spo11-Y135F::TRP1 and rec103D::KanMX (Pecina et al., 2002); sae2::LEU2 (Murakami et al., 2003). mre11D::KanMX and xrs2D::KanMX strains were obtained by transformation with PCR products containing the respective deletions amplified from strains obtained from the Yeast Deletion Project (Winzeler et al., 1999). Rad50 protein was tagged at the C terminus with 13 Myc epitopes at its original chromosomal locus by one step gene targeting (Longtine et al., 1998). In the epitope-tagged variants, no differences to the untagged yeast strains regarding MMS sensitivity, sporulation, and spore viability were detectable. Correct tagging was confirmed by Southern and Western blot analysis.
Primary data :
The primary data used in this study are here : Download raw data (Tab-delimited text file)
July 2004-Waka Lin