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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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Simulation of genetic data from sequencing analysis.A. Diagram of the DSB location and usage around ARG4, showing three prominent hotspots with relative intensities of 6, 53 and 41 (left to right) [38]. The locations of the ARG4 heteroalleles are shown; tracts that end between the alleles are capable of forming a prototroph. Examples of tracts that begin at a hotspot and end within the two heteroalleles are capable of forming an Arg+ are shown as red lines. Black lines depict examples of tracts that cannot form Arg+. B. Arg prototroph frequencies are plotted against normalized CO frequencies. Binned genetic data from Figure 3A is presented as empty symbols. Simulated values based on data from WT, HI and LO tetrads are indicated in red circles. C. The percentage of Arg prototrophs with a CO is plotted against CO frequencies. Binned genetic data from Figure 3C (empty symbols) are plotted with simulated data for WT, HI and LO tetrads (red circles). Dark blue circles represent simulations of the WT, HI and LO tract lengths but using the wild-type proportion of CO and NCO tracts. D. Same as B, with the addition of light blue circles representing mutants with 30, 10 or 3 COs as the result of extrapolating the tract length parameters (from WT, HI and LO) and performing the same simulations using an inverse regression for extrapolating the mean of the CO tract length distribution (see Materials and Methods). E. Genetic CO association data as in C, but including simulated values for virtual mutants with 30, 10 or 3 COs using an inverse regression (light blue circles) or linear relationship (green circles) (see Materials and Methods for calculations and explanation). F. Simulated CO and NCO tracts from mutants of varied Spo11 activity (WT, HI, LO, 30, 10 and 3) were scored for their ability to produce “virtual prototrophy” at ARG4.
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pgen-1003932-g005: Simulation of genetic data from sequencing analysis.A. Diagram of the DSB location and usage around ARG4, showing three prominent hotspots with relative intensities of 6, 53 and 41 (left to right) [38]. The locations of the ARG4 heteroalleles are shown; tracts that end between the alleles are capable of forming a prototroph. Examples of tracts that begin at a hotspot and end within the two heteroalleles are capable of forming an Arg+ are shown as red lines. Black lines depict examples of tracts that cannot form Arg+. B. Arg prototroph frequencies are plotted against normalized CO frequencies. Binned genetic data from Figure 3A is presented as empty symbols. Simulated values based on data from WT, HI and LO tetrads are indicated in red circles. C. The percentage of Arg prototrophs with a CO is plotted against CO frequencies. Binned genetic data from Figure 3C (empty symbols) are plotted with simulated data for WT, HI and LO tetrads (red circles). Dark blue circles represent simulations of the WT, HI and LO tract lengths but using the wild-type proportion of CO and NCO tracts. D. Same as B, with the addition of light blue circles representing mutants with 30, 10 or 3 COs as the result of extrapolating the tract length parameters (from WT, HI and LO) and performing the same simulations using an inverse regression for extrapolating the mean of the CO tract length distribution (see Materials and Methods). E. Genetic CO association data as in C, but including simulated values for virtual mutants with 30, 10 or 3 COs using an inverse regression (light blue circles) or linear relationship (green circles) (see Materials and Methods for calculations and explanation). F. Simulated CO and NCO tracts from mutants of varied Spo11 activity (WT, HI, LO, 30, 10 and 3) were scored for their ability to produce “virtual prototrophy” at ARG4.

Mentions: In order to explore whether tract length changes in the spo11 hypomorphs could account for the discrepancy between prototroph levels and crossing over, we tested if we could obtain the observed ARG4 prototroph frequencies (Figure 3A) by computationally distributing tract lengths around the ARG4 hotspot for each of the three levels of Spo11 activity. For this analysis, we took advantage of the detailed information available regarding the location and usage of meiotic DSB sites near ARG4[38]. Although these data were generated in the SK1 strain, we reasoned that the distribution of DSBs describes most genetic backgrounds since available comparisons with a hybrid strain used for microarrays and sequencing (YJM789xS96) have so far been in general agreement [7], [17], [19]. Information regarding tract length distribution and the relative abundance of NCOs and COs was used to generate sets of tracts for WT, HI and LO tetrads. Relative DSB frequencies at three hotspots in the vicinity of ARG4 were taken from a DSB hotspot map derived from Spo11-oligos and were used as a template to position these tracts [38] (Figure 5A). We assumed that a tract initiating at one of the three hotspots and ending within the region between the two ARG4 heteroalleles could generate a prototroph. We found that as Spo11 activity declined, projected prototrophs declined at an increased rate, comparable to the genetic results (Figure 5B, red dots).


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)

Simulation of genetic data from sequencing analysis.A. Diagram of the DSB location and usage around ARG4, showing three prominent hotspots with relative intensities of 6, 53 and 41 (left to right) [38]. The locations of the ARG4 heteroalleles are shown; tracts that end between the alleles are capable of forming a prototroph. Examples of tracts that begin at a hotspot and end within the two heteroalleles are capable of forming an Arg+ are shown as red lines. Black lines depict examples of tracts that cannot form Arg+. B. Arg prototroph frequencies are plotted against normalized CO frequencies. Binned genetic data from Figure 3A is presented as empty symbols. Simulated values based on data from WT, HI and LO tetrads are indicated in red circles. C. The percentage of Arg prototrophs with a CO is plotted against CO frequencies. Binned genetic data from Figure 3C (empty symbols) are plotted with simulated data for WT, HI and LO tetrads (red circles). Dark blue circles represent simulations of the WT, HI and LO tract lengths but using the wild-type proportion of CO and NCO tracts. D. Same as B, with the addition of light blue circles representing mutants with 30, 10 or 3 COs as the result of extrapolating the tract length parameters (from WT, HI and LO) and performing the same simulations using an inverse regression for extrapolating the mean of the CO tract length distribution (see Materials and Methods). E. Genetic CO association data as in C, but including simulated values for virtual mutants with 30, 10 or 3 COs using an inverse regression (light blue circles) or linear relationship (green circles) (see Materials and Methods for calculations and explanation). F. Simulated CO and NCO tracts from mutants of varied Spo11 activity (WT, HI, LO, 30, 10 and 3) were scored for their ability to produce “virtual prototrophy” at ARG4.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003932-g005: Simulation of genetic data from sequencing analysis.A. Diagram of the DSB location and usage around ARG4, showing three prominent hotspots with relative intensities of 6, 53 and 41 (left to right) [38]. The locations of the ARG4 heteroalleles are shown; tracts that end between the alleles are capable of forming a prototroph. Examples of tracts that begin at a hotspot and end within the two heteroalleles are capable of forming an Arg+ are shown as red lines. Black lines depict examples of tracts that cannot form Arg+. B. Arg prototroph frequencies are plotted against normalized CO frequencies. Binned genetic data from Figure 3A is presented as empty symbols. Simulated values based on data from WT, HI and LO tetrads are indicated in red circles. C. The percentage of Arg prototrophs with a CO is plotted against CO frequencies. Binned genetic data from Figure 3C (empty symbols) are plotted with simulated data for WT, HI and LO tetrads (red circles). Dark blue circles represent simulations of the WT, HI and LO tract lengths but using the wild-type proportion of CO and NCO tracts. D. Same as B, with the addition of light blue circles representing mutants with 30, 10 or 3 COs as the result of extrapolating the tract length parameters (from WT, HI and LO) and performing the same simulations using an inverse regression for extrapolating the mean of the CO tract length distribution (see Materials and Methods). E. Genetic CO association data as in C, but including simulated values for virtual mutants with 30, 10 or 3 COs using an inverse regression (light blue circles) or linear relationship (green circles) (see Materials and Methods for calculations and explanation). F. Simulated CO and NCO tracts from mutants of varied Spo11 activity (WT, HI, LO, 30, 10 and 3) were scored for their ability to produce “virtual prototrophy” at ARG4.
Mentions: In order to explore whether tract length changes in the spo11 hypomorphs could account for the discrepancy between prototroph levels and crossing over, we tested if we could obtain the observed ARG4 prototroph frequencies (Figure 3A) by computationally distributing tract lengths around the ARG4 hotspot for each of the three levels of Spo11 activity. For this analysis, we took advantage of the detailed information available regarding the location and usage of meiotic DSB sites near ARG4[38]. Although these data were generated in the SK1 strain, we reasoned that the distribution of DSBs describes most genetic backgrounds since available comparisons with a hybrid strain used for microarrays and sequencing (YJM789xS96) have so far been in general agreement [7], [17], [19]. Information regarding tract length distribution and the relative abundance of NCOs and COs was used to generate sets of tracts for WT, HI and LO tetrads. Relative DSB frequencies at three hotspots in the vicinity of ARG4 were taken from a DSB hotspot map derived from Spo11-oligos and were used as a template to position these tracts [38] (Figure 5A). We assumed that a tract initiating at one of the three hotspots and ending within the region between the two ARG4 heteroalleles could generate a prototroph. We found that as Spo11 activity declined, projected prototrophs declined at an increased rate, comparable to the genetic results (Figure 5B, red dots).

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