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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

Prototroph formation and CO association in spo11 hypomorphs.A. WT and spo11 mutants were sporulated at three temperatures (18°C, 22°C and 31.5°C) and plated to select Arg+. Prototroph frequencies, expressed as a percent of the maximum value observed among all strains tested at all temperatures (Table S1), are plotted against overall CO frequencies on chromosome 3, expressed as a percent of the maximum level of SPO11 COs (Table S1). The color of the data points and the associated least squares fitted lines reflects the temperature in which the diploid was sporulated (red = 31.5°C, green = 22°C and blue = 18°C and the black line encompasses data from all temperatures). B. Prototrophs were measured at LEU2 for three spo11 mutants (alleles 217, 179 and 32) and WT at three temperatures. Additional points (31.5°C) represent three Spo11 “reduced” mutants (see Table S3, S6). Raw data were normalized as described above and then plotted. The solid black line shows the least squares fit for the complete dataset. C. WT and spo11 mutants were sporulated at 18°C, 22°C and 31.5°C (blue, green and red data points, respectively) and assayed for flanking marker exchange among Arg prototrophs (Table S1). The colored lines reflect the least squares fit for each temperature and the solid black line represents a least squares fit to the complete dataset. The % CO association @ ARG4 is the percent of Arg prototrophs with a CO in the TRP1-THR1 interval. D. Three mutants (alleles 217, 179 and 32) and three Spo11 “reduced” mutants (see Table S3, S6) and WT were assayed for CO association at LEU2. The percent of Leu prototrophs with a CO in the HIS4-iHYG@CEN interval was plotted against overall CO values. The solid black line represents a least squares fit to the data. The formula used for the least squares fit is a second order polynomial.
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pgen-1003932-g003: Prototroph formation and CO association in spo11 hypomorphs.A. WT and spo11 mutants were sporulated at three temperatures (18°C, 22°C and 31.5°C) and plated to select Arg+. Prototroph frequencies, expressed as a percent of the maximum value observed among all strains tested at all temperatures (Table S1), are plotted against overall CO frequencies on chromosome 3, expressed as a percent of the maximum level of SPO11 COs (Table S1). The color of the data points and the associated least squares fitted lines reflects the temperature in which the diploid was sporulated (red = 31.5°C, green = 22°C and blue = 18°C and the black line encompasses data from all temperatures). B. Prototrophs were measured at LEU2 for three spo11 mutants (alleles 217, 179 and 32) and WT at three temperatures. Additional points (31.5°C) represent three Spo11 “reduced” mutants (see Table S3, S6). Raw data were normalized as described above and then plotted. The solid black line shows the least squares fit for the complete dataset. C. WT and spo11 mutants were sporulated at 18°C, 22°C and 31.5°C (blue, green and red data points, respectively) and assayed for flanking marker exchange among Arg prototrophs (Table S1). The colored lines reflect the least squares fit for each temperature and the solid black line represents a least squares fit to the complete dataset. The % CO association @ ARG4 is the percent of Arg prototrophs with a CO in the TRP1-THR1 interval. D. Three mutants (alleles 217, 179 and 32) and three Spo11 “reduced” mutants (see Table S3, S6) and WT were assayed for CO association at LEU2. The percent of Leu prototrophs with a CO in the HIS4-iHYG@CEN interval was plotted against overall CO values. The solid black line represents a least squares fit to the data. The formula used for the least squares fit is a second order polynomial.

Mentions: If prototroph formation and crossing over are similarly impacted by decreased Spo11 activity, then a directly proportional relationship should be observed when CO frequencies are plotted against prototroph frequencies. However, prototroph formation is affected more severely than crossing over (Figure 3A) and this trend is apparent in the entire spectrum of mutants and temperatures. The disparity between crossover and gene conversion levels increased as Spo11 activity declined. For example, a mutant with less than 1% of the wild-type level of prototroph formation exhibits as much as 15% of the wild-type level of crossing over (e.g., spo11-179 at 18°C, Table S1). This is seen even when mutants with greater than 20% of wild-type CO levels are plotted by temperature i.e. the three regression lines would have a positive X intercept rather than at zero. The use of spo13 dyads to generate spores avoids a viability bias in these analyses. However, if there is significant heterogeneity on a cell-to-cell basis for recombination within a genotype, it is possible that the observed disparity of CO and prototroph frequencies may be somewhat exaggerated. A similar trend to Arg prototrophy was observed when prototroph formation was measured at the LEU2 locus on chromosome 3, using a subset of SPO11 hypomorphs (Figure 3B, Table S4).


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)

Prototroph formation and CO association in spo11 hypomorphs.A. WT and spo11 mutants were sporulated at three temperatures (18°C, 22°C and 31.5°C) and plated to select Arg+. Prototroph frequencies, expressed as a percent of the maximum value observed among all strains tested at all temperatures (Table S1), are plotted against overall CO frequencies on chromosome 3, expressed as a percent of the maximum level of SPO11 COs (Table S1). The color of the data points and the associated least squares fitted lines reflects the temperature in which the diploid was sporulated (red = 31.5°C, green = 22°C and blue = 18°C and the black line encompasses data from all temperatures). B. Prototrophs were measured at LEU2 for three spo11 mutants (alleles 217, 179 and 32) and WT at three temperatures. Additional points (31.5°C) represent three Spo11 “reduced” mutants (see Table S3, S6). Raw data were normalized as described above and then plotted. The solid black line shows the least squares fit for the complete dataset. C. WT and spo11 mutants were sporulated at 18°C, 22°C and 31.5°C (blue, green and red data points, respectively) and assayed for flanking marker exchange among Arg prototrophs (Table S1). The colored lines reflect the least squares fit for each temperature and the solid black line represents a least squares fit to the complete dataset. The % CO association @ ARG4 is the percent of Arg prototrophs with a CO in the TRP1-THR1 interval. D. Three mutants (alleles 217, 179 and 32) and three Spo11 “reduced” mutants (see Table S3, S6) and WT were assayed for CO association at LEU2. The percent of Leu prototrophs with a CO in the HIS4-iHYG@CEN interval was plotted against overall CO values. The solid black line represents a least squares fit to the data. The formula used for the least squares fit is a second order polynomial.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003932-g003: Prototroph formation and CO association in spo11 hypomorphs.A. WT and spo11 mutants were sporulated at three temperatures (18°C, 22°C and 31.5°C) and plated to select Arg+. Prototroph frequencies, expressed as a percent of the maximum value observed among all strains tested at all temperatures (Table S1), are plotted against overall CO frequencies on chromosome 3, expressed as a percent of the maximum level of SPO11 COs (Table S1). The color of the data points and the associated least squares fitted lines reflects the temperature in which the diploid was sporulated (red = 31.5°C, green = 22°C and blue = 18°C and the black line encompasses data from all temperatures). B. Prototrophs were measured at LEU2 for three spo11 mutants (alleles 217, 179 and 32) and WT at three temperatures. Additional points (31.5°C) represent three Spo11 “reduced” mutants (see Table S3, S6). Raw data were normalized as described above and then plotted. The solid black line shows the least squares fit for the complete dataset. C. WT and spo11 mutants were sporulated at 18°C, 22°C and 31.5°C (blue, green and red data points, respectively) and assayed for flanking marker exchange among Arg prototrophs (Table S1). The colored lines reflect the least squares fit for each temperature and the solid black line represents a least squares fit to the complete dataset. The % CO association @ ARG4 is the percent of Arg prototrophs with a CO in the TRP1-THR1 interval. D. Three mutants (alleles 217, 179 and 32) and three Spo11 “reduced” mutants (see Table S3, S6) and WT were assayed for CO association at LEU2. The percent of Leu prototrophs with a CO in the HIS4-iHYG@CEN interval was plotted against overall CO values. The solid black line represents a least squares fit to the data. The formula used for the least squares fit is a second order polynomial.
Mentions: If prototroph formation and crossing over are similarly impacted by decreased Spo11 activity, then a directly proportional relationship should be observed when CO frequencies are plotted against prototroph frequencies. However, prototroph formation is affected more severely than crossing over (Figure 3A) and this trend is apparent in the entire spectrum of mutants and temperatures. The disparity between crossover and gene conversion levels increased as Spo11 activity declined. For example, a mutant with less than 1% of the wild-type level of prototroph formation exhibits as much as 15% of the wild-type level of crossing over (e.g., spo11-179 at 18°C, Table S1). This is seen even when mutants with greater than 20% of wild-type CO levels are plotted by temperature i.e. the three regression lines would have a positive X intercept rather than at zero. The use of spo13 dyads to generate spores avoids a viability bias in these analyses. However, if there is significant heterogeneity on a cell-to-cell basis for recombination within a genotype, it is possible that the observed disparity of CO and prototroph frequencies may be somewhat exaggerated. A similar trend to Arg prototrophy was observed when prototroph formation was measured at the LEU2 locus on chromosome 3, using a subset of SPO11 hypomorphs (Figure 3B, Table S4).

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