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High throughput sequencing reveals alterations in the recombination signatures with diminishing Spo11 activity.

Rockmill B, Lefrançois P, Voelkel-Meiman K, Oke A, Roeder GS, Fung JC - PLoS Genet. (2013)

Bottom Line: Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants.High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type.Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.

ABSTRACT
Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

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Related in: MedlinePlus

Tract-Seq and summary of sequence data.A. Tract-Seq analysis to generate an estimated tract length, given maximal and minimal tracts. The red rectangle depicts the minimal tract length (“core”) in which reflects a contiguous stretch of 1∶3 markers and blue rectangles on either side of the core represent the regions in which the tract endpoints must be located. (i) The enzymatic resection machinery has processed through the minimal tract. (ii) Assuming a bidirectional enzyme with a symmetric enzymatic processivity, the probability of the enzyme falling off on either side of the core region follows a decreasing exponential law. (iii) Enzymatic complexes (green balls), initially located at the edges of the core region, fall off independently on each side of the core. (iv) The enzymes fall off before reaching the boundaries of the maximal tract. The estimated tract spans the distance between the two endpoints, encompassing the core interval. B. Analysis of wild-type tracts. Left: Histograms of midpoint-estimated (grey bars) and Tract-Seq-estimated (black-outlined transparent bars) individual CO (top) and NCO (bottom) tract lengths. Right: Histograms of Tract-Seq-estimated individual CO (top) and NCO (bottom) tract lengths, with the overlaid best-fitted log-normal distributions of GC tract lengths for each case. C. Histograms of Tract-Seq-estimated (black-outlined transparent bars) of individual NCO (left) and CO (right) tract lengths, along with the overlaid best-fitted log-normal distributions of GC tract lengths for Hi (top) and LO (bottom) mutants. D and E. Tract-Seq was used to estimate tract lengths for GC events associated with COs (D) and for NCOs (E). The probabilities of tracts of different lengths are plotted against tract length (in base pairs). Simulations based on WT (black line), HI (red line) and LO (blue line) tetrads are graphed, and respective means (dotted lines) are shown. Means and 95% confidence intervals are presented in Table 2. A log normal distribution was used to fit the distribution of tract lengths. The mean and standard deviation are the parameters needed to define each log normal distribution.
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pgen-1003932-g004: Tract-Seq and summary of sequence data.A. Tract-Seq analysis to generate an estimated tract length, given maximal and minimal tracts. The red rectangle depicts the minimal tract length (“core”) in which reflects a contiguous stretch of 1∶3 markers and blue rectangles on either side of the core represent the regions in which the tract endpoints must be located. (i) The enzymatic resection machinery has processed through the minimal tract. (ii) Assuming a bidirectional enzyme with a symmetric enzymatic processivity, the probability of the enzyme falling off on either side of the core region follows a decreasing exponential law. (iii) Enzymatic complexes (green balls), initially located at the edges of the core region, fall off independently on each side of the core. (iv) The enzymes fall off before reaching the boundaries of the maximal tract. The estimated tract spans the distance between the two endpoints, encompassing the core interval. B. Analysis of wild-type tracts. Left: Histograms of midpoint-estimated (grey bars) and Tract-Seq-estimated (black-outlined transparent bars) individual CO (top) and NCO (bottom) tract lengths. Right: Histograms of Tract-Seq-estimated individual CO (top) and NCO (bottom) tract lengths, with the overlaid best-fitted log-normal distributions of GC tract lengths for each case. C. Histograms of Tract-Seq-estimated (black-outlined transparent bars) of individual NCO (left) and CO (right) tract lengths, along with the overlaid best-fitted log-normal distributions of GC tract lengths for Hi (top) and LO (bottom) mutants. D and E. Tract-Seq was used to estimate tract lengths for GC events associated with COs (D) and for NCOs (E). The probabilities of tracts of different lengths are plotted against tract length (in base pairs). Simulations based on WT (black line), HI (red line) and LO (blue line) tetrads are graphed, and respective means (dotted lines) are shown. Means and 95% confidence intervals are presented in Table 2. A log normal distribution was used to fit the distribution of tract lengths. The mean and standard deviation are the parameters needed to define each log normal distribution.

Mentions: In order to better define tract lengths, we developed an algorithm, called Tract-Seq, that estimates tract length using a Markov model for tract growth combined with Monte Carlo simulations (Materials and Methods, Figure 4A). Figure 4B shows the distribution of wild-type tract lengths calculated by Tract-Seq in comparison to the distribution calculated by taking the midpoints. We generated similar graphs of “Tract-Seq” lengths for HI and LO (Figure 4C). Average tract lengths were determined from the log-normal fits for the distribution of CO-associated tracts and NCO tracts for WT, HI and LO tetrads (Figure 4D and E). CO-associated tracts increase in length as the Spo11 activity decreases; the means increased by 56 bp for HI tetrads and 316 bp for LO tetrads, compared to WT (Table 2). However, CO-associated tract lengths for LO tetrads, but not HI tetrads, compared to WT are significantly longer (p = 0.05 for LO vs. WT and p = 0.28 for HI vs. WT). In contrast, NCO tracts are smaller in context of decreased Spo11 activity; the mean length of tracts decreased by 134 for HI and 292 bp for LO (Table 2). Again, the difference in NCO tract lengths relative to WT is significant for LO tetrads, but not for HI tetrads (p = 0.02 for LO vs. WT and p = 0.17 for HI vs. WT). For both CO- and NCO-associated tracts, the range of possible tract lengths increases as Spo11 activity decreases.


High throughput sequencing reveals alterations in the recombination signatures with diminishing Spo11 activity.

Rockmill B, Lefrançois P, Voelkel-Meiman K, Oke A, Roeder GS, Fung JC - PLoS Genet. (2013)

Tract-Seq and summary of sequence data.A. Tract-Seq analysis to generate an estimated tract length, given maximal and minimal tracts. The red rectangle depicts the minimal tract length (“core”) in which reflects a contiguous stretch of 1∶3 markers and blue rectangles on either side of the core represent the regions in which the tract endpoints must be located. (i) The enzymatic resection machinery has processed through the minimal tract. (ii) Assuming a bidirectional enzyme with a symmetric enzymatic processivity, the probability of the enzyme falling off on either side of the core region follows a decreasing exponential law. (iii) Enzymatic complexes (green balls), initially located at the edges of the core region, fall off independently on each side of the core. (iv) The enzymes fall off before reaching the boundaries of the maximal tract. The estimated tract spans the distance between the two endpoints, encompassing the core interval. B. Analysis of wild-type tracts. Left: Histograms of midpoint-estimated (grey bars) and Tract-Seq-estimated (black-outlined transparent bars) individual CO (top) and NCO (bottom) tract lengths. Right: Histograms of Tract-Seq-estimated individual CO (top) and NCO (bottom) tract lengths, with the overlaid best-fitted log-normal distributions of GC tract lengths for each case. C. Histograms of Tract-Seq-estimated (black-outlined transparent bars) of individual NCO (left) and CO (right) tract lengths, along with the overlaid best-fitted log-normal distributions of GC tract lengths for Hi (top) and LO (bottom) mutants. D and E. Tract-Seq was used to estimate tract lengths for GC events associated with COs (D) and for NCOs (E). The probabilities of tracts of different lengths are plotted against tract length (in base pairs). Simulations based on WT (black line), HI (red line) and LO (blue line) tetrads are graphed, and respective means (dotted lines) are shown. Means and 95% confidence intervals are presented in Table 2. A log normal distribution was used to fit the distribution of tract lengths. The mean and standard deviation are the parameters needed to define each log normal distribution.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814317&req=5

pgen-1003932-g004: Tract-Seq and summary of sequence data.A. Tract-Seq analysis to generate an estimated tract length, given maximal and minimal tracts. The red rectangle depicts the minimal tract length (“core”) in which reflects a contiguous stretch of 1∶3 markers and blue rectangles on either side of the core represent the regions in which the tract endpoints must be located. (i) The enzymatic resection machinery has processed through the minimal tract. (ii) Assuming a bidirectional enzyme with a symmetric enzymatic processivity, the probability of the enzyme falling off on either side of the core region follows a decreasing exponential law. (iii) Enzymatic complexes (green balls), initially located at the edges of the core region, fall off independently on each side of the core. (iv) The enzymes fall off before reaching the boundaries of the maximal tract. The estimated tract spans the distance between the two endpoints, encompassing the core interval. B. Analysis of wild-type tracts. Left: Histograms of midpoint-estimated (grey bars) and Tract-Seq-estimated (black-outlined transparent bars) individual CO (top) and NCO (bottom) tract lengths. Right: Histograms of Tract-Seq-estimated individual CO (top) and NCO (bottom) tract lengths, with the overlaid best-fitted log-normal distributions of GC tract lengths for each case. C. Histograms of Tract-Seq-estimated (black-outlined transparent bars) of individual NCO (left) and CO (right) tract lengths, along with the overlaid best-fitted log-normal distributions of GC tract lengths for Hi (top) and LO (bottom) mutants. D and E. Tract-Seq was used to estimate tract lengths for GC events associated with COs (D) and for NCOs (E). The probabilities of tracts of different lengths are plotted against tract length (in base pairs). Simulations based on WT (black line), HI (red line) and LO (blue line) tetrads are graphed, and respective means (dotted lines) are shown. Means and 95% confidence intervals are presented in Table 2. A log normal distribution was used to fit the distribution of tract lengths. The mean and standard deviation are the parameters needed to define each log normal distribution.
Mentions: In order to better define tract lengths, we developed an algorithm, called Tract-Seq, that estimates tract length using a Markov model for tract growth combined with Monte Carlo simulations (Materials and Methods, Figure 4A). Figure 4B shows the distribution of wild-type tract lengths calculated by Tract-Seq in comparison to the distribution calculated by taking the midpoints. We generated similar graphs of “Tract-Seq” lengths for HI and LO (Figure 4C). Average tract lengths were determined from the log-normal fits for the distribution of CO-associated tracts and NCO tracts for WT, HI and LO tetrads (Figure 4D and E). CO-associated tracts increase in length as the Spo11 activity decreases; the means increased by 56 bp for HI tetrads and 316 bp for LO tetrads, compared to WT (Table 2). However, CO-associated tract lengths for LO tetrads, but not HI tetrads, compared to WT are significantly longer (p = 0.05 for LO vs. WT and p = 0.28 for HI vs. WT). In contrast, NCO tracts are smaller in context of decreased Spo11 activity; the mean length of tracts decreased by 134 for HI and 292 bp for LO (Table 2). Again, the difference in NCO tract lengths relative to WT is significant for LO tetrads, but not for HI tetrads (p = 0.02 for LO vs. WT and p = 0.17 for HI vs. WT). For both CO- and NCO-associated tracts, the range of possible tract lengths increases as Spo11 activity decreases.

Bottom Line: Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants.High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type.Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America.

ABSTRACT
Spo11 is the topoisomerase-like enzyme responsible for the induction of the meiosis-specific double strand breaks (DSBs), which initiates the recombination events responsible for proper chromosome segregation. Nineteen PCR-induced alleles of SPO11 were identified and characterized genetically and cytologically. Recombination, spore viability and synaptonemal complex (SC) formation were decreased to varying extents in these mutants. Arrest by ndt80 restored these events in two severe hypomorphic mutants, suggesting that ndt80-arrested nuclei are capable of extended DSB activity. While crossing-over, spore viability and synaptonemal complex (SC) formation defects correlated, the extent of such defects was not predictive of the level of heteroallelic gene conversions (prototrophs) exhibited by each mutant. High throughput sequencing of tetrads from spo11 hypomorphs revealed that gene conversion tracts associated with COs are significantly longer and gene conversion tracts unassociated with COs are significantly shorter than in wild type. By modeling the extent of these tract changes, we could account for the discrepancy in genetic measurements of prototrophy and crossover association. These findings provide an explanation for the unexpectedly low prototroph levels exhibited by spo11 hypomorphs and have important implications for genetic studies that assume an unbiased recovery of prototrophs, such as measurements of CO homeostasis. Our genetic and physical data support previous observations of DSB-limited meioses, in which COs are disproportionally maintained over NCOs (CO homeostasis).

Show MeSH
Related in: MedlinePlus