Limits...
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).

Show MeSH

Related in: MedlinePlus

Pachytene arrest rescues spo11 mutants.A. Extent of SC formed in spo11 mutants in NDT80+ and ndt80 backgrounds after 48 hours sporulation at 18°C. White bars are partial SC (i.e., at least two linear tracts of SC, “some SC” in Table S2) and dark bars represent nuclei with most chromatin containing Zip1 in stretches (“Full SC” in Table S2). At least 100 spreads exhibiting Red1 staining were scored for each mutant. B. Arg prototrophs were measured in “return-to-growth” assays from at least 4 cultures (Table S1) and the average is plotted with standard deviations. C and D. gNDT80 strains (pGal-NDT80 – GAL4.ER) were arrested at pachytene for 48 hours and then induced to sporulate by the addition of estrogen. C. Spore viability was scored among 85 tetrads for pGal-NDT80, 172 tetrads for spo11-217 pGal-NDT80 and 25 tetrads for spo11-179 pGal-NDT80. D. At least 100 four-spore-viable tetrads were scored for recombination for three intervals on chromosome 3. Control NDT80+ data was from tetrad dissection and spo11-217 data is from isolated spo13 spores (Table S1, S3).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814317&req=5

pgen-1003932-g002: Pachytene arrest rescues spo11 mutants.A. Extent of SC formed in spo11 mutants in NDT80+ and ndt80 backgrounds after 48 hours sporulation at 18°C. White bars are partial SC (i.e., at least two linear tracts of SC, “some SC” in Table S2) and dark bars represent nuclei with most chromatin containing Zip1 in stretches (“Full SC” in Table S2). At least 100 spreads exhibiting Red1 staining were scored for each mutant. B. Arg prototrophs were measured in “return-to-growth” assays from at least 4 cultures (Table S1) and the average is plotted with standard deviations. C and D. gNDT80 strains (pGal-NDT80 – GAL4.ER) were arrested at pachytene for 48 hours and then induced to sporulate by the addition of estrogen. C. Spore viability was scored among 85 tetrads for pGal-NDT80, 172 tetrads for spo11-217 pGal-NDT80 and 25 tetrads for spo11-179 pGal-NDT80. D. At least 100 four-spore-viable tetrads were scored for recombination for three intervals on chromosome 3. Control NDT80+ data was from tetrad dissection and spo11-217 data is from isolated spo13 spores (Table S1, S3).

Mentions: In order to score synapsis in a uniformly arrested population for several severely defective spo11 hypomorphs, we measured SC formation in an ndt80 background. Ndt80 promotes progression beyond the pachytene stage of meiosis, thus SPO11 ndt80 cells arrest with fully synapsed chromosomes [34]. Interestingly, while some severe spo11 mutants only produced rare nuclei with partial SCs, in the ndt80 background such mutants displayed up to 96% of meiotic chromosome spreads with SC (Figure 2A, Table S2). This ndt80-associated restoration of synapsis was apparent for several alleles (179, 217, 245, 1025 and 240, Table S2 and data not shown) but either weakly restored or absent in other alleles (246, 117 and 845, data not shown). To further examine this apparent rescue of the spo11 phenotype, recombination levels were measured by scoring Arg prototroph formation after 48 hours of ndt80-arrest by a return to growth assay. While previous work reported small increases in heteroallelic prototrophy levels in ndt80- and cdc28-arrested cells [34], [35] our WT ndt80 strain did not increase Arg prototrophy levels compared to WT (Figure 2). However, Arg prototroph formation was increased approximately 10 and 60-fold in spo11-179 ndt80 and spo11-217 ndt80, respectively (Figure 2B, Table S1) compared to the mutants in the NDT80 background, and reached about 10% of WT (and SPO11 ndt80) levels.


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)

Pachytene arrest rescues spo11 mutants.A. Extent of SC formed in spo11 mutants in NDT80+ and ndt80 backgrounds after 48 hours sporulation at 18°C. White bars are partial SC (i.e., at least two linear tracts of SC, “some SC” in Table S2) and dark bars represent nuclei with most chromatin containing Zip1 in stretches (“Full SC” in Table S2). At least 100 spreads exhibiting Red1 staining were scored for each mutant. B. Arg prototrophs were measured in “return-to-growth” assays from at least 4 cultures (Table S1) and the average is plotted with standard deviations. C and D. gNDT80 strains (pGal-NDT80 – GAL4.ER) were arrested at pachytene for 48 hours and then induced to sporulate by the addition of estrogen. C. Spore viability was scored among 85 tetrads for pGal-NDT80, 172 tetrads for spo11-217 pGal-NDT80 and 25 tetrads for spo11-179 pGal-NDT80. D. At least 100 four-spore-viable tetrads were scored for recombination for three intervals on chromosome 3. Control NDT80+ data was from tetrad dissection and spo11-217 data is from isolated spo13 spores (Table S1, S3).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003932-g002: Pachytene arrest rescues spo11 mutants.A. Extent of SC formed in spo11 mutants in NDT80+ and ndt80 backgrounds after 48 hours sporulation at 18°C. White bars are partial SC (i.e., at least two linear tracts of SC, “some SC” in Table S2) and dark bars represent nuclei with most chromatin containing Zip1 in stretches (“Full SC” in Table S2). At least 100 spreads exhibiting Red1 staining were scored for each mutant. B. Arg prototrophs were measured in “return-to-growth” assays from at least 4 cultures (Table S1) and the average is plotted with standard deviations. C and D. gNDT80 strains (pGal-NDT80 – GAL4.ER) were arrested at pachytene for 48 hours and then induced to sporulate by the addition of estrogen. C. Spore viability was scored among 85 tetrads for pGal-NDT80, 172 tetrads for spo11-217 pGal-NDT80 and 25 tetrads for spo11-179 pGal-NDT80. D. At least 100 four-spore-viable tetrads were scored for recombination for three intervals on chromosome 3. Control NDT80+ data was from tetrad dissection and spo11-217 data is from isolated spo13 spores (Table S1, S3).
Mentions: In order to score synapsis in a uniformly arrested population for several severely defective spo11 hypomorphs, we measured SC formation in an ndt80 background. Ndt80 promotes progression beyond the pachytene stage of meiosis, thus SPO11 ndt80 cells arrest with fully synapsed chromosomes [34]. Interestingly, while some severe spo11 mutants only produced rare nuclei with partial SCs, in the ndt80 background such mutants displayed up to 96% of meiotic chromosome spreads with SC (Figure 2A, Table S2). This ndt80-associated restoration of synapsis was apparent for several alleles (179, 217, 245, 1025 and 240, Table S2 and data not shown) but either weakly restored or absent in other alleles (246, 117 and 845, data not shown). To further examine this apparent rescue of the spo11 phenotype, recombination levels were measured by scoring Arg prototroph formation after 48 hours of ndt80-arrest by a return to growth assay. While previous work reported small increases in heteroallelic prototrophy levels in ndt80- and cdc28-arrested cells [34], [35] our WT ndt80 strain did not increase Arg prototrophy levels compared to WT (Figure 2). However, Arg prototroph formation was increased approximately 10 and 60-fold in spo11-179 ndt80 and spo11-217 ndt80, respectively (Figure 2B, Table S1) compared to the mutants in the NDT80 background, and reached about 10% of WT (and SPO11 ndt80) levels.

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