Borde and colleagues reported that a reporter plasmid inserted at different genomic locations in Saccharomyces cerevisiae had different levels of meiotic recombination activity. We show that the level of recombination activity is very significantly correlated with the GC content of DNA sequences flanking the insertion

Merker, Jason D.

Petes, Thomas D.

PubMed

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Copyright  2002 by the Genetics Society of AmericaNoteContext Dependence of Meiotic Recombination Hotspots in Yeast:The Relationship Between Recombination Activity of aReporter Construct and Base CompositionThomas D. Petes1 and Jason D. MerkerDepartment of Biology and Curriculum in Genetics and Molecular Biology, University of North Carolina,Chapel Hill, North Carolina 27599-3280Manuscript received August 17, 2002Accepted for publication September 3, 2002ABSTRACTBorde and colleagues reported that a reporter plasmid inserted at different genomic locations in Saccharomycescerevisiae had different levels of meiotic recombination activity. We show that the level of recombinationactivity is very significantly correlated with the GC content of DNA sequences flanking the insertion.IN eukaryotes, chromosomal regions of high (hotspots) genomic hotspots to single open reading frame resolu-and low (coldspots) meiotic recombination activity tion and found many hotspots were associated with localhave been identified (Lichten and Goldman 1995; regions of high GC content. One interpretation of thesePetes 2001). In Saccharomyces cerevisiae, the recombina- results is that GC-rich intergenic regions are a preferredtion activity of a chromosomal region is primarily a substrate for the recombination machinery.function of the frequency of local double-stranded DNA Borde et al. (1999) monitored recombination eventsbreaks (DSBs). DSBs generally occur between genes in within reporter plasmids (Figure 1) introduced into 10nuclease-sensitive chromatin (Wu and Lichten 1994). different locations on yeast chromosome III. Two differ-Three different types of hotspots (not necessarily mutu- ent assays of recombination activity were performed.ally exclusive) have been described. The activity of -hot- First, Borde and colleagues measured the frequency ofspots requires transcription factor binding, but not tran- DSBs located at two positions within the inserted plas-scription per se (White et al. 1992, 1993). There are also mid, indicated as DSB-left and DSB-right in Figure 1.hotspots associated with constitutively nucleosome-free Second, they determined the frequency of heteroallelicregions of DNA (; Kirkpatrick et al. 1999) and local recombination between the two plasmids (pMJ113 andregions of high G  C base composition (; Gerton pMJ115) placed at allelic positions. In general, theseet al. 2000). The mechanisms responsible for hotspot two measurements gave the same relative strength ofactivity are not understood, although it has been sug- recombination activity. Borde et al. (1999) found thatgested that all hotspots may be in regions with hyper- recombination activity, as measured by either of thesemodified nucleosomes (Petes 2001). assays, varied by more than a factor of 10, dependingThe relationship between naturally occurring hot- on the position of the insertion.spots in S. cerevisiae and chromosomal regions of high To determine whether the recombination activity ofG  C base composition has been examined in several the plasmid insertions in the study of Borde et al. (1999)studies. Sharp and Lloyd (1993) found that chromo- was correlated with the G  C content of DNA sequencessome III had two broad (50 kb) regions of high G  flanking the insertion, we used the “Composition” pro-C content and pointed out that three of the four known gram of GCG to analyze the G  C content in variousrecombination hotspots were located in the GC-rich “windows” of flanking DNA for all 10 insertions, varyingregions. Baudat and Nicolas (1997) mapped all ofthe size of the window from 500 bp (250 bp to eachthe DSB sites on chromosome III, demonstrating thatside of the insertion) to 100 kb (50 kb to each side ofmost of the strong DSB sites were located in these twothe insertion). For each window, the correlation betweenregions. Gerton et al. (2001) used microarrays to mapG  C content and the recombination activity (as mea-sured by the sum of the DSBs at DSB-left and DSB-right)was determined using both the Pearson parametric test1Corresponding author: Department of Biology, University of North and the Spearman nonparametric test. For almost allCarolina, Chapel Hill, NC 27599-3280.E-mail: tompetes@email.unc.edu windows, the P values of the correlation were highlyGenetics 162: 2049–2052 (December 2002)2050 T. D. Petes and J. D. MerkerFigure 1.—Plasmids used to monitor recombination activ-ity. The 8.5-kb plasmids (a) pMJ113 and (b) pMJ115 containpBR322 sequences and the URA3 and ARG4 genes (Borde etal. 1999). Plasmids pMJ113 and pMJ115 contain the mutantalleles arg4-nsp and arg4-Bgl, respectively. Genomic DNA frag- Figure 2.—The correlation between recombination activityments derived from yeast chromosome III were inserted into in the reporter constructs (measured by the frequency of DSBthese plasmids, and the resulting plasmid derivatives were formation) and the GC content in a 100-kb window. Bordeintegrated into the chromosomes of haploid a and -strains; et al. (1999) measured recombination activity of the reporterthese haploid strains were subsequently crossed to generate plasmids inserted at 10 positions on chromosome III. Onediploids. Meiotic recombination was measured in two ways measurement of this activity was the sum of the DSB frequency(Borde et al. 1999). In RAD50 diploids, the frequency of Arg for both DSB-left and DSB-right. In this graph, this parameterspores, resulting from heteroallelic exchange between arg4- is shown as a function of the GC content of the flankingnsp and arg4-Bgl, was measured. In rad50S diploids, Southern genomic sequences, measured 50 kb to each side of the inser-analysis was used to measure the frequency of DSB formation tion (100-kb window); since one of the plasmid insertions (atat the positions indicated by arrows. CHA1) was located 50 kb from the telomere, this insertionwas not included in the analysis. The insert locations, the “%DSB” and the “% G  C” content for the nine other insertions,significant (Table 1). The correlation between DSB ac- are: HIS4 (B, 16.5% DSB, 39.2% GC); LEU2 (C, 14.2% DSB,tivity and the flanking G  C content (100-kb window) 39.3% GC); YCL011c (D, 10.6% DSB, 38.5% GC); YCR004c(E, 2.8% DSB, 37.7% GC); RVS161 (F, 1.9% DSB, 37.2% GC);for nine insertions is shown in Figure 2. The positionsYCR017c (G, 2.8% DSB, 37.2% GC); YCR026c (H, 4.3% DSB,37.4% GC); RIM1 (I, 5.4% DSB, 37.8% GC); MAT (J, 10.2%DSB, 38.6% GC).TABLE 1Correlation coefficients (r values) and the associated probabilitiesof correlations (P values) for the relationship of G  C of the insertions in relation to base composition forcontent (measured in windows from 0.5 to 100 kb)windows of 5 and 100 kb are shown in Figure 3.and the frequency of meiotic recombinationIn Table 1, the P values for correlations determined(assayed by double-strand break formation)with the Pearson test (which assumes a linear correla-tion) fall into two groups. For windows between 0.5 andPearson test Spearman testWindow size 20 kb, the P values are between 0.005 and 0.02; for(kb) r values P values r values P values windows between 30 and 100 kb, the P values are allsubstantially lower, between 0.0001 and 0.001. This0.5 0.808 0.005 0.857 0.0021 0.639 0.05 0.657 0.04 analysis indicates that the recombination activity of in-2 0.774 0.009 0.809 0.005 serted sequences is better predicted by the GC content2.5 0.760 0.01 0.809 0.005 of large chromosomal regions (30 kb) than by that5 0.750 0.01 0.681 0.03of smaller regions (20 kb). The P values for the non-10 0.778 0.008 0.686 0.03parametric Spearman test did not follow the same sim-20 0.709 0.02 0.511 0.1330 0.930 0.0001 0.726 0.02 ple pattern. Since the correlation coefficients (r values)40 0.894 0.001 0.789 0.01 obtained with the Pearson test for the large chromo-50 0.904 0.0008 0.765 0.02 somal regions are larger than those obtained with the60 0.888 0.001 0.702 0.04Spearman test for any window (except for the 100-kb75 0.934 0.0002 0.740 0.02window), it is likely that the Pearson test represents the100 0.961 0.0001 0.929 0.0003better method of analyzing the data.For each window, the correlation between G  C contentFrom our analysis, we suggest two conclusions. First,and the recombination activity (as measured by the sum ofthe context dependence of the recombination proper-the DSBs at DSB-left and DSB-right) was determined usingthe SAS JMP software (version 4.0.4; SAS Institute, Cary, NC); ties of plasmid insertions observed by Borde et al. (1999)we determined correlation coefficients (r values) and their reflects, at least in part, the base composition of theassociated probabilities (P values) using both the Pearson DNA sequences flanking the insertion. These effects actparametric test and the Spearman nonparametric test. Sinceat a distance, since the DSB sites used to assay recombi-one of the insertions (near the CHA1 gene) is 15 kb fromnation are located 1 kb from the junction with thethe telomere, this insertion was not used to calculate r and Pvalues for windows 30 kb. chromosomal DNA. Second, our analysis supports the2051NoteFigure 3.—Base composition of chromo-some III assayed using moving windows of5 and 100 kb. (a) 5-kb moving window: GCcontent was determined (using the “Win-dow” Program of GCG) in a window of 5kb moved in 1-kb intervals. The arrows withletters show the positions of the insertions(A represents the CHA1 insertion; code forB–J in Figure 2). The short arrows at thetop of the figure indicate nine peaks ofG  C that are 3% above the average forchromosome III (38.7%). The numberedarrows represent hotspots for recombina-tion as determined by Gerton et al. (2000).(b) 100-kb moving window: GC content wasdetermined in windows of 100 kb moved in1-kb intervals.earlier conclusion (Sharp and Lloyd 1993; Baudat Galtier et al. 2001; Marais et al. 2001), since highrecombination rates result in elevated levels of hetero-and Nicolas 1997) that there are recombination-stimu-duplex formation and since mismatches in hetero-lating effects of high G  C that involve large (30 kb)duplexes are usually repaired with a bias toward GCchromosomal regions. On the basis of the colocalizationover AT in mitotic mammalian and yeast cells (Brownof hotspots and peaks of local (5 kb) G  C contentand Jiricny 1989; Birdsell 2002) and in meiotic yeast(Gerton et al. 2000; Birdsell 2002), there appear tocells (Birdsell 2002). The first model is more consis-be both local and regional effects of G  C contenttent with the observation that GC-rich sequences de-on meiotic recombination. Local effects could reflect arived from Escherichia coli plasmids often have unusuallypreference of the recombination machinery to associatestrong hotspot activity when introduced into the yeastwith high-GC intergenic regions whereas regional ef-genome (Stapleton and Petes 1991; Wu and Lichtenfects may represent some global feature of chromosome1995), although (as discussed above) the hotspot activitystructure that is affected by base composition (Zicklerof these sequences is influenced by the GC content ofand Kleckner 1999); such global features could bethe chromosomal DNA sequences that flank the inser-regions of recombination-promoting hyperacetylatedtion.nucleosomes or recombination-suppressing hypermeth-Although our results and those of others support theylated nucleosomes. The reporter plasmids were GCconclusion that GC content affects hotspot activity, localrich (46.2%) compared to yeast genomic DNA (39%)and regional GC content is only one factor. In S. cerevis-and therefore would be expected to contribute to bothiae, other factors associated with hotspot activity include:local and regional GC richness. Since this contribution(1) “open” chromatin, (2) a location between diver-is the same for each position of integration, the relativegently transcribed genes, and (3) a requirement forrecombination activities of the insertions should be in-local transcription factor binding (Lichten and Gold-dependent of this effect.man 1995; Petes 2001); hotspots are found infrequentlyA correlation between high recombination activityin centromeric or telomeric regions (Baudat and Nico-and high GC content has also been observed for humanslas 1997; Gerton et al. 2000). Although these associa-(Eisenbarth et al. 2001), Caenorhabditis elegans, and Dro-tions have some predictive value in determining whichsophila melanogaster (Marais et al. 2001). Two differentDNA sequences will be recombination hotspots, thetypes of explanation have been given for this relation-mechanisms responsible for these associations are notship. We suggested that regions of high GC stimulateunderstood.recombination (Gerton et al. 2000; Petes 2001). Alter-natively, high levels of recombination may create geno- We thank P. Mieczkowski, J. Stone, and M. Lichten for useful discus-sions and J. Stone, M. A. Amamoo, D. Moore, and N. Degtyarevamic regions with high GC content (Eyre-Walker 1993;2052 T. D. Petes and J. D. Merkerexpression in yeast by a simple repetitive DNA sequence thatfor help with data analysis. The research was supported by Nationalexcludes nucleosomes. Mol. Cell. Biol. 19: 7661–7671.Institutes of Health grant GM-24110.Lichten, M., and A. S. H. Goldman, 1995 Meiotic recombinationhotspots. Annu. Rev. Genet. 29: 423–444.Marais, G., D. Mouchiroud and L. Duret, 2001 Does recombina-tion improve selection on codon usage? Lessons from nematodeLITERATURE CITEDand fly complete genomes. Proc. Natl. Acad. Sci. USA 98: 5688–Baudat, F., and A. Nicolas, 1997 Clustering of meiotic double- 5692.strand breaks on yeast chromosome III. Proc. Natl. Acad. Sci. Petes, T. D., 2001 Meiotic recombination hot spots and cold spots.USA 94: 5213–5218. Nat. Rev. Genet. 2: 360–369.Birdsell, J. A., 2002 Integrating genomics, bioinformatics, and clas- Sharp, P. M., and A. T. Lloyd, 1993 Regional base compositionsical genetics to study the effects of recombination on genome variation along yeast chromosome III: evolution of chromosomeevolution. Mol. Biol. Evol. 19: 1181–1197. primary structure. Nucleic Acids Res. 21: 179–183.Borde, V., T.-C. Wu and M. Lichten, 1999 Use of a recombination Stapleton, A., and T. D. 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D. Griffith andT. D. Petes, 1999 Control of meiotic recombination and gene Communicating editor: A. Nicolas

Context dependence of meiotic recombination hotspots in yeast: the relationship between recombination activity of a reporter construct and base composition.

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Context dependence of meiotic recombination hotspots in yeast: the relationship between recombination activity of a reporter construct and base composition.

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