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required for strand invasion in homologous recombination, RAD52 (Casper et al. 2009). In oour studies, class 3-5 profiles were observed following the depletion of NSE1 and UBC9, which also function in DNA repair (Branzei et al 2006; Santa Maria et al. 2007; Irmisch et al 2009). We found one addi- tional class 3-5 rearrangement following the depletion of DPB11 (Table 2). In each case, the breakpoint on chromosome III corresponds with the only Ty retrotransposon on the left arm and the rearrangement results in the loss of one copy of the chromosome III centromere. We predict that this acentric chromosome III fragment would be fused to the nonhomolo- gous chromosome to allow this chromosome fragment to per- sist (Figure 5B). The chromosome fusion could result from an interruption ofa BIR event and subsequent resolution of the strand invasion intermediate with a nonhomologous chromo- some, resulting in a half crossover chromosome. Alternatively since the breakpoints on the nonhomologous chromosomes coincide with Ty retrotransposon sites, it is possible that a ho- mology-mediated repair mechanism, such as single-strand annealing (SSA) (Mieczkowski et al. 2006), is involved in the formation of this chromosome fusion (Figure 5B). This mutant chromosome was confirmed by Southerrn blot analysis of a representative illegitimate diploid isolated following the depletion of NSE1 (Figure 6C, lanes 2 and 4). A probe specific for chromosome III detected two chromosome sizes, one cor- responding to intact chromosome III and another to the pre- dicted size of the chromosome III-chromosome V fusion. We also detected two chromosome bands following rehybrid- ization with a probe specific for chromosome V, one cor- responding to the size of intact chromosome V and the other corresponding to the expected size of the chromosome fusion. Finally, depletion of RFC2 resulted in one example of a "Haw thorne deletion" (class 3-6), an interstitial deletion between repeated regions of the MATa and HMRa loci (Hawthorne 1963). We did not observe any examples of class 3-7, the hallmark of which is amplification of sequences between FS1 and FS2 (Casper et al. 2009). The total ratio of the seven rearrangement classes was 15:4:8:4:6:1:0 (3-1: 3-2: 3-3: 3-4: 3-5: 3-6: 3-7). XV Class 3-3 3-4 3-1 3-2 3-2 3-5 3-5 Figure 6 Southern blot confirmation of chromosome rearrangements predicted in microarray analysis. (A) Intact chromosomal DNA was iso- lated from class 3 (chromosome arm loss) illegitimate diploids. Genomic DNA was separated on a contour-clamped homogenous electric field (CHEF) gel and chromosome IIl was detected through hybridization with a radio-labeled probe specific to the left arm of chromosome lII. Smaller chromosome ll fragments were also detected in samples with chromo- some rearrangements. Representative Southern blots for a wild-type dip- loid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown, respectively. (B) Representative Southern blots of a wild-type and a class 3-2 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosomes IIl and XV, respectively. In addition to chromosome IlIl and XV, a nonreciprocal translocation (nrt) was visualized. (C) Representative Southern blot of a wild-type and a class 3-5 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosome Ill and V, respectively. In addition to chromosomes II and V, a chromosome fusion (fus) event was detected. support the indicated structure of this class of rearrangement (Figure 5B) Class 3-4 illegitimate diploids have only a deletion of the right arm of chromosome III (Figures 4D and 5B). We ob- served four of these events after the depletion of genes in- volved in DNA replication (MCM7, DPB11, and SPT16). Two of these events had breakpoints at FS1 and FS2 (MCM7 and DPB11), the same breakpoints observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). The other two, from SPT16 illegitimate diploids, had break points at YCRC86. As suggested previously, this class might represent chromosome fragments that persist through direct telomere capping (Figure 5B) or by acquisition of telomeric sequences by BIR utilizing a telomere-proximal Ty element (Lemoine et al. 2005, 2008). These rearrangement profiles were confirmed by Southern blot analysis, where two bands of equal intensity were visualized, one corresponding to in tact chromosome III and the other to the predicted chromo- some III fragment (Figure 6A, lane 3) The class 3-5 rearrangement pattern includes a deletion of all but the left arm of chromosome III in addition to an arm deletion on a nonhomologous chromosome (chromosome XVI or V) (Figures 4E and 5B). By contrast to the four sub- classes of rearrangements described above, class 3-5 profiles were not observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). However, this profile has been documented upon the deletion of a nonessential gene Boundaries of rearrangements correlate with Ty retrotransposons, LTRS, tRNA genes, early replication origins, and replication termination sites To determine whether the boundaries of rearrangements are correlated with particular genomic features, we performed an enrichment test. We segmented the genome into 5-kb bins and scored each bin for the presence or absence of genomic features and breakpoints. For each feature, we determined the fold enrichment of bins that contain both the feature and a breakpoint in comparison to what would be expected if breakpoints were randomly placed into bins (Table 3). Con- sistent with other studies of rearrangement breakpoints in yeast (Dunham et al. 2002; Lemoine et al. 2005, 2008 Argueso et al. 2008; Li et al. 2009), our boundaries of re- arrangement were significantly enriched at loci with Ty retro- transposons, LTRS, and tRNA genes (Table 3). As these sites Essential Genome Stability Genes 155 SPT16-2-1-1 DPB11-1-1-2 MCM7-2-1-1 WT SPT16-1-3-2 SPT16-1-3-2 NSE1-4-5-1 NSE1-4-5-1

required for strand invasion in homologous recombination, RAD52 (Casper et al. 2009). In oour studies, class 3-5 profiles were observed following the depletion of NSE1 and UBC9, which also function in DNA repair (Branzei et al 2006; Santa Maria et al. 2007; Irmisch et al 2009). We found one addi- tional class 3-5 rearrangement following the depletion of DPB11 (Table 2). In each case, the breakpoint on chromosome III corresponds with the only Ty retrotransposon on the left arm and the rearrangement results in the loss of one copy of the chromosome III centromere. We predict that this acentric chromosome III fragment would be fused to the nonhomolo- gous chromosome to allow this chromosome fragment to per- sist (Figure 5B). The chromosome fusion could result from an interruption ofa BIR event and subsequent resolution of the strand invasion intermediate with a nonhomologous chromo- some, resulting in a half crossover chromosome. Alternatively since the breakpoints on the nonhomologous chromosomes coincide with Ty retrotransposon sites, it is possible that a ho- mology-mediated repair mechanism, such as single-strand annealing (SSA) (Mieczkowski et al. 2006), is involved in the formation of this chromosome fusion (Figure 5B). This mutant chromosome was confirmed by Southerrn blot analysis of a representative illegitimate diploid isolated following the depletion of NSE1 (Figure 6C, lanes 2 and 4). A probe specific for chromosome III detected two chromosome sizes, one cor- responding to intact chromosome III and another to the pre- dicted size of the chromosome III-chromosome V fusion. We also detected two chromosome bands following rehybrid- ization with a probe specific for chromosome V, one cor- responding to the size of intact chromosome V and the other corresponding to the expected size of the chromosome fusion. Finally, depletion of RFC2 resulted in one example of a "Haw thorne deletion" (class 3-6), an interstitial deletion between repeated regions of the MATa and HMRa loci (Hawthorne 1963). We did not observe any examples of class 3-7, the hallmark of which is amplification of sequences between FS1 and FS2 (Casper et al. 2009). The total ratio of the seven rearrangement classes was 15:4:8:4:6:1:0 (3-1: 3-2: 3-3: 3-4: 3-5: 3-6: 3-7). XV Class 3-3 3-4 3-1 3-2 3-2 3-5 3-5 Figure 6 Southern blot confirmation of chromosome rearrangements predicted in microarray analysis. (A) Intact chromosomal DNA was iso- lated from class 3 (chromosome arm loss) illegitimate diploids. Genomic DNA was separated on a contour-clamped homogenous electric field (CHEF) gel and chromosome IIl was detected through hybridization with a radio-labeled probe specific to the left arm of chromosome lII. Smaller chromosome ll fragments were also detected in samples with chromo- some rearrangements. Representative Southern blots for a wild-type dip- loid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown, respectively. (B) Representative Southern blots of a wild-type and a class 3-2 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosomes IIl and XV, respectively. In addition to chromosome IlIl and XV, a nonreciprocal translocation (nrt) was visualized. (C) Representative Southern blot of a wild-type and a class 3-5 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosome Ill and V, respectively. In addition to chromosomes II and V, a chromosome fusion (fus) event was detected. support the indicated structure of this class of rearrangement (Figure 5B) Class 3-4 illegitimate diploids have only a deletion of the right arm of chromosome III (Figures 4D and 5B). We ob- served four of these events after the depletion of genes in- volved in DNA replication (MCM7, DPB11, and SPT16). Two of these events had breakpoints at FS1 and FS2 (MCM7 and DPB11), the same breakpoints observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). The other two, from SPT16 illegitimate diploids, had break points at YCRC86. As suggested previously, this class might represent chromosome fragments that persist through direct telomere capping (Figure 5B) or by acquisition of telomeric sequences by BIR utilizing a telomere-proximal Ty element (Lemoine et al. 2005, 2008). These rearrangement profiles were confirmed by Southern blot analysis, where two bands of equal intensity were visualized, one corresponding to in tact chromosome III and the other to the predicted chromo- some III fragment (Figure 6A, lane 3) The class 3-5 rearrangement pattern includes a deletion of all but the left arm of chromosome III in addition to an arm deletion on a nonhomologous chromosome (chromosome XVI or V) (Figures 4E and 5B). By contrast to the four sub- classes of rearrangements described above, class 3-5 profiles were not observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). However, this profile has been documented upon the deletion of a nonessential gene Boundaries of rearrangements correlate with Ty retrotransposons, LTRS, tRNA genes, early replication origins, and replication termination sites To determine whether the boundaries of rearrangements are correlated with particular genomic features, we performed an enrichment test. We segmented the genome into 5-kb bins and scored each bin for the presence or absence of genomic features and breakpoints. For each feature, we determined the fold enrichment of bins that contain both the feature and a breakpoint in comparison to what would be expected if breakpoints were randomly placed into bins (Table 3). Con- sistent with other studies of rearrangement breakpoints in yeast (Dunham et al. 2002; Lemoine et al. 2005, 2008 Argueso et al. 2008; Li et al. 2009), our boundaries of re- arrangement were significantly enriched at loci with Ty retro- transposons, LTRS, and tRNA genes (Table 3). As these sites Essential Genome Stability Genes 155 SPT16-2-1-1 DPB11-1-1-2 MCM7-2-1-1 WT SPT16-1-3-2 SPT16-1-3-2 NSE1-4-5-1 NSE1-4-5-1

Biochemistry
Biochemistry
6th Edition
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Reginald H. Garrett, Charles M. Grisham
Chapter28: Dna Metabolism: Replication, Recombination, And Repair
Section: Chapter Questions
Problem 10P: Homologous Recombination, Heteroduplex DNA, and Mismatch Repair Homologous recombination in E. coli...
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What is Boundaries of rearrangements correlate with Ty retrotransposons. LTRs, tRNA genes, early replication origins, and replication termination sites major result mean in this experiment? (interpretation)

required for strand invasion in homologous recombination, RAD52 (Casper et al. 2009). In oour studies, class 3-5 profiles were observed following the depletion of NSE1 and UBC9, which also function in DNA repair (Branzei et al 2006; Santa Maria et al. 2007; Irmisch et al 2009). We found one addi- tional class 3-5 rearrangement following the depletion of DPB11 (Table 2). In each case, the breakpoint on chromosome III corresponds with the only Ty retrotransposon on the left arm and the rearrangement results in the loss of one copy of the chromosome III centromere. We predict that this acentric chromosome III fragment would be fused to the nonhomolo- gous chromosome to allow this chromosome fragment to per- sist (Figure 5B). The chromosome fusion could result from an interruption ofa BIR event and subsequent resolution of the strand invasion intermediate with a nonhomologous chromo- some, resulting in a half crossover chromosome. Alternatively since the breakpoints on the nonhomologous chromosomes coincide with Ty retrotransposon sites, it is possible that a ho- mology-mediated repair mechanism, such as single-strand annealing (SSA) (Mieczkowski et al. 2006), is involved in the formation of this chromosome fusion (Figure 5B). This mutant chromosome was confirmed by Southerrn blot analysis of a representative illegitimate diploid isolated following the depletion of NSE1 (Figure 6C, lanes 2 and 4). A probe specific for chromosome III detected two chromosome sizes, one cor- responding to intact chromosome III and another to the pre- dicted size of the chromosome III-chromosome V fusion. We also detected two chromosome bands following rehybrid- ization with a probe specific for chromosome V, one cor- responding to the size of intact chromosome V and the other corresponding to the expected size of the chromosome fusion. Finally, depletion of RFC2 resulted in one example of a "Haw thorne deletion" (class 3-6), an interstitial deletion between repeated regions of the MATa and HMRa loci (Hawthorne 1963). We did not observe any examples of class 3-7, the hallmark of which is amplification of sequences between FS1 and FS2 (Casper et al. 2009). The total ratio of the seven rearrangement classes was 15:4:8:4:6:1:0 (3-1: 3-2: 3-3: 3-4: 3-5: 3-6: 3-7). XV Class 3-3 3-4 3-1 3-2 3-2 3-5 3-5 Figure 6 Southern blot confirmation of chromosome rearrangements predicted in microarray analysis. (A) Intact chromosomal DNA was iso- lated from class 3 (chromosome arm loss) illegitimate diploids. Genomic DNA was separated on a contour-clamped homogenous electric field (CHEF) gel and chromosome IIl was detected through hybridization with a radio-labeled probe specific to the left arm of chromosome lII. Smaller chromosome ll fragments were also detected in samples with chromo- some rearrangements. Representative Southern blots for a wild-type dip- loid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown, respectively. (B) Representative Southern blots of a wild-type and a class 3-2 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosomes IIl and XV, respectively. In addition to chromosome IlIl and XV, a nonreciprocal translocation (nrt) was visualized. (C) Representative Southern blot of a wild-type and a class 3-5 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosome Ill and V, respectively. In addition to chromosomes II and V, a chromosome fusion (fus) event was detected. support the indicated structure of this class of rearrangement (Figure 5B) Class 3-4 illegitimate diploids have only a deletion of the right arm of chromosome III (Figures 4D and 5B). We ob- served four of these events after the depletion of genes in- volved in DNA replication (MCM7, DPB11, and SPT16). Two of these events had breakpoints at FS1 and FS2 (MCM7 and DPB11), the same breakpoints observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). The other two, from SPT16 illegitimate diploids, had break points at YCRC86. As suggested previously, this class might represent chromosome fragments that persist through direct telomere capping (Figure 5B) or by acquisition of telomeric sequences by BIR utilizing a telomere-proximal Ty element (Lemoine et al. 2005, 2008). These rearrangement profiles were confirmed by Southern blot analysis, where two bands of equal intensity were visualized, one corresponding to in tact chromosome III and the other to the predicted chromo- some III fragment (Figure 6A, lane 3) The class 3-5 rearrangement pattern includes a deletion of all but the left arm of chromosome III in addition to an arm deletion on a nonhomologous chromosome (chromosome XVI or V) (Figures 4E and 5B). By contrast to the four sub- classes of rearrangements described above, class 3-5 profiles were not observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). However, this profile has been documented upon the deletion of a nonessential gene Boundaries of rearrangements correlate with Ty retrotransposons, LTRS, tRNA genes, early replication origins, and replication termination sites To determine whether the boundaries of rearrangements are correlated with particular genomic features, we performed an enrichment test. We segmented the genome into 5-kb bins and scored each bin for the presence or absence of genomic features and breakpoints. For each feature, we determined the fold enrichment of bins that contain both the feature and a breakpoint in comparison to what would be expected if breakpoints were randomly placed into bins (Table 3). Con- sistent with other studies of rearrangement breakpoints in yeast (Dunham et al. 2002; Lemoine et al. 2005, 2008 Argueso et al. 2008; Li et al. 2009), our boundaries of re- arrangement were significantly enriched at loci with Ty retro- transposons, LTRS, and tRNA genes (Table 3). As these sites Essential Genome Stability Genes 155 SPT16-2-1-1 DPB11-1-1-2 MCM7-2-1-1 WT SPT16-1-3-2 SPT16-1-3-2 NSE1-4-5-1 NSE1-4-5-1
Transcribed Image Text:required for strand invasion in homologous recombination, RAD52 (Casper et al. 2009). In oour studies, class 3-5 profiles were observed following the depletion of NSE1 and UBC9, which also function in DNA repair (Branzei et al 2006; Santa Maria et al. 2007; Irmisch et al 2009). We found one addi- tional class 3-5 rearrangement following the depletion of DPB11 (Table 2). In each case, the breakpoint on chromosome III corresponds with the only Ty retrotransposon on the left arm and the rearrangement results in the loss of one copy of the chromosome III centromere. We predict that this acentric chromosome III fragment would be fused to the nonhomolo- gous chromosome to allow this chromosome fragment to per- sist (Figure 5B). The chromosome fusion could result from an interruption ofa BIR event and subsequent resolution of the strand invasion intermediate with a nonhomologous chromo- some, resulting in a half crossover chromosome. Alternatively since the breakpoints on the nonhomologous chromosomes coincide with Ty retrotransposon sites, it is possible that a ho- mology-mediated repair mechanism, such as single-strand annealing (SSA) (Mieczkowski et al. 2006), is involved in the formation of this chromosome fusion (Figure 5B). This mutant chromosome was confirmed by Southerrn blot analysis of a representative illegitimate diploid isolated following the depletion of NSE1 (Figure 6C, lanes 2 and 4). A probe specific for chromosome III detected two chromosome sizes, one cor- responding to intact chromosome III and another to the pre- dicted size of the chromosome III-chromosome V fusion. We also detected two chromosome bands following rehybrid- ization with a probe specific for chromosome V, one cor- responding to the size of intact chromosome V and the other corresponding to the expected size of the chromosome fusion. Finally, depletion of RFC2 resulted in one example of a "Haw thorne deletion" (class 3-6), an interstitial deletion between repeated regions of the MATa and HMRa loci (Hawthorne 1963). We did not observe any examples of class 3-7, the hallmark of which is amplification of sequences between FS1 and FS2 (Casper et al. 2009). The total ratio of the seven rearrangement classes was 15:4:8:4:6:1:0 (3-1: 3-2: 3-3: 3-4: 3-5: 3-6: 3-7). XV Class 3-3 3-4 3-1 3-2 3-2 3-5 3-5 Figure 6 Southern blot confirmation of chromosome rearrangements predicted in microarray analysis. (A) Intact chromosomal DNA was iso- lated from class 3 (chromosome arm loss) illegitimate diploids. Genomic DNA was separated on a contour-clamped homogenous electric field (CHEF) gel and chromosome IIl was detected through hybridization with a radio-labeled probe specific to the left arm of chromosome lII. Smaller chromosome ll fragments were also detected in samples with chromo- some rearrangements. Representative Southern blots for a wild-type dip- loid, classes 3-3, 3-4, and 3-1 diploids of the indicated strains are shown, respectively. (B) Representative Southern blots of a wild-type and a class 3-2 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosomes IIl and XV, respectively. In addition to chromosome IlIl and XV, a nonreciprocal translocation (nrt) was visualized. (C) Representative Southern blot of a wild-type and a class 3-5 illegitimate diploid. Chromosomes on the left and right were detected with probes for chromosome Ill and V, respectively. In addition to chromosomes II and V, a chromosome fusion (fus) event was detected. support the indicated structure of this class of rearrangement (Figure 5B) Class 3-4 illegitimate diploids have only a deletion of the right arm of chromosome III (Figures 4D and 5B). We ob- served four of these events after the depletion of genes in- volved in DNA replication (MCM7, DPB11, and SPT16). Two of these events had breakpoints at FS1 and FS2 (MCM7 and DPB11), the same breakpoints observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). The other two, from SPT16 illegitimate diploids, had break points at YCRC86. As suggested previously, this class might represent chromosome fragments that persist through direct telomere capping (Figure 5B) or by acquisition of telomeric sequences by BIR utilizing a telomere-proximal Ty element (Lemoine et al. 2005, 2008). These rearrangement profiles were confirmed by Southern blot analysis, where two bands of equal intensity were visualized, one corresponding to in tact chromosome III and the other to the predicted chromo- some III fragment (Figure 6A, lane 3) The class 3-5 rearrangement pattern includes a deletion of all but the left arm of chromosome III in addition to an arm deletion on a nonhomologous chromosome (chromosome XVI or V) (Figures 4E and 5B). By contrast to the four sub- classes of rearrangements described above, class 3-5 profiles were not observed following depletion of DNA polymerases a and 8 (Lemoine et al. 2005, 2008). However, this profile has been documented upon the deletion of a nonessential gene Boundaries of rearrangements correlate with Ty retrotransposons, LTRS, tRNA genes, early replication origins, and replication termination sites To determine whether the boundaries of rearrangements are correlated with particular genomic features, we performed an enrichment test. We segmented the genome into 5-kb bins and scored each bin for the presence or absence of genomic features and breakpoints. For each feature, we determined the fold enrichment of bins that contain both the feature and a breakpoint in comparison to what would be expected if breakpoints were randomly placed into bins (Table 3). Con- sistent with other studies of rearrangement breakpoints in yeast (Dunham et al. 2002; Lemoine et al. 2005, 2008 Argueso et al. 2008; Li et al. 2009), our boundaries of re- arrangement were significantly enriched at loci with Ty retro- transposons, LTRS, and tRNA genes (Table 3). As these sites Essential Genome Stability Genes 155 SPT16-2-1-1 DPB11-1-1-2 MCM7-2-1-1 WT SPT16-1-3-2 SPT16-1-3-2 NSE1-4-5-1 NSE1-4-5-1
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