Limits...
Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

Goldfarb T, Lichten M - PLoS Biol. (2010)

Bottom Line: This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies.These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids.We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.

Show MeSH

Related in: MedlinePlus

Altered DSB and JM metabolism in mek1Δ strains.(A) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3201 and MJL3198); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3250 and MJL3338); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3370 and MJL3381). Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis; MJL3201, MJL3250, and MJL3370); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains (right-hand y-axis; MJL3198, MJL3338, and MJL3381). (B) Cumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from ndt80Δ MEK1 and ndt80Δ mek1Δ strains. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3252); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3497); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3387). (C) Noncumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from the same RAD50 strains used for DSB analysis in (A). (D and E) Timing of the meiosis I (D) and meiosis II (E) nuclear divisions, monitored by DAPI staining (see Protocol S1; 3–4 independent experiments, error bars indicate standard deviation), in the same RAD50 strains used for DSB and JM analysis in (A) and (C).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2957403&req=5

pbio-1000520-g004: Altered DSB and JM metabolism in mek1Δ strains.(A) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3201 and MJL3198); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3250 and MJL3338); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3370 and MJL3381). Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis; MJL3201, MJL3250, and MJL3370); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains (right-hand y-axis; MJL3198, MJL3338, and MJL3381). (B) Cumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from ndt80Δ MEK1 and ndt80Δ mek1Δ strains. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3252); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3497); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3387). (C) Noncumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from the same RAD50 strains used for DSB analysis in (A). (D and E) Timing of the meiosis I (D) and meiosis II (E) nuclear divisions, monitored by DAPI staining (see Protocol S1; 3–4 independent experiments, error bars indicate standard deviation), in the same RAD50 strains used for DSB and JM analysis in (A) and (C).

Mentions: DSBs form at normal levels but are more rapidly repaired in strains lacking Mek1 kinase activity, as compared to wild-type [17],[19],[21]–[23],[42]. A similar decrease in DSB life span is seen in cells with an unphosphorylatable Hop1 protein that does not activate the Mek1 kinase [18]. Because mek1 strains also show greatly reduced IH recombination [21],[23],[42],[43], it has been suggested that, in the absence of Mek1 activity, meiotic DSBs are rapidly repaired by IS recombination. We confirmed that, in mek1Δ strains, steady-state DSB levels are substantially reduced at a hemizygous his4::URA3-arg4 insert and at a homozygous YCR047c site, while cumulative DSB levels, measured in rad50S strains, are not affected (Figure 4A). Thus, DSB life spans are substantially reduced in mek1Δ relative to wild-type (by about 3-fold; data not shown). Because DSBs in hemizygous loci are repaired by IS recombination, this indicates that loss of Mek1 increases the rate of IS recombination by about a factor of three.


Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis.

Goldfarb T, Lichten M - PLoS Biol. (2010)

Altered DSB and JM metabolism in mek1Δ strains.(A) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3201 and MJL3198); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3250 and MJL3338); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3370 and MJL3381). Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis; MJL3201, MJL3250, and MJL3370); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains (right-hand y-axis; MJL3198, MJL3338, and MJL3381). (B) Cumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from ndt80Δ MEK1 and ndt80Δ mek1Δ strains. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3252); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3497); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3387). (C) Noncumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from the same RAD50 strains used for DSB analysis in (A). (D and E) Timing of the meiosis I (D) and meiosis II (E) nuclear divisions, monitored by DAPI staining (see Protocol S1; 3–4 independent experiments, error bars indicate standard deviation), in the same RAD50 strains used for DSB and JM analysis in (A) and (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957403&req=5

pbio-1000520-g004: Altered DSB and JM metabolism in mek1Δ strains.(A) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3201 and MJL3198); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3250 and MJL3338); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3370 and MJL3381). Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis; MJL3201, MJL3250, and MJL3370); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains (right-hand y-axis; MJL3198, MJL3338, and MJL3381). (B) Cumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from ndt80Δ MEK1 and ndt80Δ mek1Δ strains. Symbols: blue diamonds, fully homozygous MEK1 strain (MJL3252); green squares, MEK1 strains hemizygous for his4::URA3-arg4 (MJL3497); and pink circles, mek1Δ strain hemizygous for his4::URA3-arg4 (MJL3387). (C) Noncumulative JM frequencies (3–4 independent experiments, error bars indicate SEM) from the same RAD50 strains used for DSB analysis in (A). (D and E) Timing of the meiosis I (D) and meiosis II (E) nuclear divisions, monitored by DAPI staining (see Protocol S1; 3–4 independent experiments, error bars indicate standard deviation), in the same RAD50 strains used for DSB and JM analysis in (A) and (C).
Mentions: DSBs form at normal levels but are more rapidly repaired in strains lacking Mek1 kinase activity, as compared to wild-type [17],[19],[21]–[23],[42]. A similar decrease in DSB life span is seen in cells with an unphosphorylatable Hop1 protein that does not activate the Mek1 kinase [18]. Because mek1 strains also show greatly reduced IH recombination [21],[23],[42],[43], it has been suggested that, in the absence of Mek1 activity, meiotic DSBs are rapidly repaired by IS recombination. We confirmed that, in mek1Δ strains, steady-state DSB levels are substantially reduced at a hemizygous his4::URA3-arg4 insert and at a homozygous YCR047c site, while cumulative DSB levels, measured in rad50S strains, are not affected (Figure 4A). Thus, DSB life spans are substantially reduced in mek1Δ relative to wild-type (by about 3-fold; data not shown). Because DSBs in hemizygous loci are repaired by IS recombination, this indicates that loss of Mek1 increases the rate of IS recombination by about a factor of three.

Bottom Line: This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies.These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids.We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.

Show MeSH
Related in: MedlinePlus