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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.

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(A) Timing of molecular events at a hemizygous (MJL3250) and homozygous (MJL3201) his4::URA3-arg4 insert. Left- and right-hand edges of rectangles indicate half-maximum points on cumulative curves for formation and repair/resolution, respectively. For meiotic divisions, left- and right-hand edges indicate 50% times for meiosis I (M1) and meiosis II (M2), respectively. Times are normalized by setting the 50% time for meiosis II to 6 h (actual times ± standard deviation: MJL3201, 5.66±0.27 h; MJL3250, 6.08±0.33 h). Left- and right-hand error bars denote the standard deviation for the half-maximum value and the sum of the standard deviations of the life span and the half-maximum value, respectively. (B) Estimation of IS/IH ratio for all recombination events at homozygous loci in wild-type, based on the following: (1) about 1/2 of IH events involve JMs; (2) about 1/4 to 1/6 of IS events involve JMs; (3) about 1/5 of JMs are IS (numbers in parentheses are observed range in the literature). Based on these values, an IS/IH total event ratio of 1∶1.7 to 1∶2.5 is calculated. Detailed calculations and IS/IH total event ratios for the full range of IS/IH JM ratios are in Protocol S1 and Figure S2. (C) How localized kinase activation can cause selective retardation of IS recombination. DSBs form when potential DSB sites on cohesed sister chromatids (pink boxes) are recruited to the chromosome axis (green). Mec1/Tel1 checkpoint kinases are activated by DSBs and associated single-stranded DNA, and phosphorylate chromatin and axis proteins in the vicinity of DSBs (red). Phosphorylated axis proteins recruit and activate Mek1 kinase, which phosphorylates target proteins (including strand transferase accessory proteins) in the vicinity of the DSB-activated axis. Strand invasion of the sister chromatid, which is within the zone of axis-associated inhibition, is thus kinetically impeded; strand invasion of the homolog is unaffected.
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pbio-1000520-g006: (A) Timing of molecular events at a hemizygous (MJL3250) and homozygous (MJL3201) his4::URA3-arg4 insert. Left- and right-hand edges of rectangles indicate half-maximum points on cumulative curves for formation and repair/resolution, respectively. For meiotic divisions, left- and right-hand edges indicate 50% times for meiosis I (M1) and meiosis II (M2), respectively. Times are normalized by setting the 50% time for meiosis II to 6 h (actual times ± standard deviation: MJL3201, 5.66±0.27 h; MJL3250, 6.08±0.33 h). Left- and right-hand error bars denote the standard deviation for the half-maximum value and the sum of the standard deviations of the life span and the half-maximum value, respectively. (B) Estimation of IS/IH ratio for all recombination events at homozygous loci in wild-type, based on the following: (1) about 1/2 of IH events involve JMs; (2) about 1/4 to 1/6 of IS events involve JMs; (3) about 1/5 of JMs are IS (numbers in parentheses are observed range in the literature). Based on these values, an IS/IH total event ratio of 1∶1.7 to 1∶2.5 is calculated. Detailed calculations and IS/IH total event ratios for the full range of IS/IH JM ratios are in Protocol S1 and Figure S2. (C) How localized kinase activation can cause selective retardation of IS recombination. DSBs form when potential DSB sites on cohesed sister chromatids (pink boxes) are recruited to the chromosome axis (green). Mec1/Tel1 checkpoint kinases are activated by DSBs and associated single-stranded DNA, and phosphorylate chromatin and axis proteins in the vicinity of DSBs (red). Phosphorylated axis proteins recruit and activate Mek1 kinase, which phosphorylates target proteins (including strand transferase accessory proteins) in the vicinity of the DSB-activated axis. Strand invasion of the sister chromatid, which is within the zone of axis-associated inhibition, is thus kinetically impeded; strand invasion of the homolog is unaffected.

Mentions: Our genetic and molecular data indicate that the sister chromatid can be used as efficiently as the homolog in the repair of meiotic DSBs. DSBs that form at hemizygous loci are repaired with the same efficiency and timing as DSBs formed at homozygous loci (Figures 1 and 6A). While these DSBs could, in theory, be repaired by IH gene conversion of the entire region of heterology, such events are relatively rare (Figure 2). We therefore conclude that the majority of DSBs that form at hemizygous loci are repaired by recombination between sister chromatids. Furthermore, the efficient repair of DSBs that form opposite deletions of an entire chromosome arm (Figure 1) indicates that nearby IH interactions are not required for IS recombination to occur. While repair in hemizygous strains occurs exclusively from the sister chromatid, Hunter and colleagues have suggested that multiple templates, including the sister chromatid, are frequently used in the repair of DSBs when both parental homologs are present [11]. JMs containing three and four chromatids form in wild-type cells, and are abundant in strains lacking the Sgs1 helicase [11]. This supports the suggestions that multiple repair templates are often used during meiotic recombination, that recombination is a dynamic process, and that Sgs1 acts to prevent aberrant structures that are formed as a result of these processes [11],[50]. The increased incidence of IS JMs in strains lacking Sgs1 further supports the claim that the sister chromatid is often used for DSB repair during meiosis [11],[12],[49].


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)

(A) Timing of molecular events at a hemizygous (MJL3250) and homozygous (MJL3201) his4::URA3-arg4 insert. Left- and right-hand edges of rectangles indicate half-maximum points on cumulative curves for formation and repair/resolution, respectively. For meiotic divisions, left- and right-hand edges indicate 50% times for meiosis I (M1) and meiosis II (M2), respectively. Times are normalized by setting the 50% time for meiosis II to 6 h (actual times ± standard deviation: MJL3201, 5.66±0.27 h; MJL3250, 6.08±0.33 h). Left- and right-hand error bars denote the standard deviation for the half-maximum value and the sum of the standard deviations of the life span and the half-maximum value, respectively. (B) Estimation of IS/IH ratio for all recombination events at homozygous loci in wild-type, based on the following: (1) about 1/2 of IH events involve JMs; (2) about 1/4 to 1/6 of IS events involve JMs; (3) about 1/5 of JMs are IS (numbers in parentheses are observed range in the literature). Based on these values, an IS/IH total event ratio of 1∶1.7 to 1∶2.5 is calculated. Detailed calculations and IS/IH total event ratios for the full range of IS/IH JM ratios are in Protocol S1 and Figure S2. (C) How localized kinase activation can cause selective retardation of IS recombination. DSBs form when potential DSB sites on cohesed sister chromatids (pink boxes) are recruited to the chromosome axis (green). Mec1/Tel1 checkpoint kinases are activated by DSBs and associated single-stranded DNA, and phosphorylate chromatin and axis proteins in the vicinity of DSBs (red). Phosphorylated axis proteins recruit and activate Mek1 kinase, which phosphorylates target proteins (including strand transferase accessory proteins) in the vicinity of the DSB-activated axis. Strand invasion of the sister chromatid, which is within the zone of axis-associated inhibition, is thus kinetically impeded; strand invasion of the homolog is unaffected.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000520-g006: (A) Timing of molecular events at a hemizygous (MJL3250) and homozygous (MJL3201) his4::URA3-arg4 insert. Left- and right-hand edges of rectangles indicate half-maximum points on cumulative curves for formation and repair/resolution, respectively. For meiotic divisions, left- and right-hand edges indicate 50% times for meiosis I (M1) and meiosis II (M2), respectively. Times are normalized by setting the 50% time for meiosis II to 6 h (actual times ± standard deviation: MJL3201, 5.66±0.27 h; MJL3250, 6.08±0.33 h). Left- and right-hand error bars denote the standard deviation for the half-maximum value and the sum of the standard deviations of the life span and the half-maximum value, respectively. (B) Estimation of IS/IH ratio for all recombination events at homozygous loci in wild-type, based on the following: (1) about 1/2 of IH events involve JMs; (2) about 1/4 to 1/6 of IS events involve JMs; (3) about 1/5 of JMs are IS (numbers in parentheses are observed range in the literature). Based on these values, an IS/IH total event ratio of 1∶1.7 to 1∶2.5 is calculated. Detailed calculations and IS/IH total event ratios for the full range of IS/IH JM ratios are in Protocol S1 and Figure S2. (C) How localized kinase activation can cause selective retardation of IS recombination. DSBs form when potential DSB sites on cohesed sister chromatids (pink boxes) are recruited to the chromosome axis (green). Mec1/Tel1 checkpoint kinases are activated by DSBs and associated single-stranded DNA, and phosphorylate chromatin and axis proteins in the vicinity of DSBs (red). Phosphorylated axis proteins recruit and activate Mek1 kinase, which phosphorylates target proteins (including strand transferase accessory proteins) in the vicinity of the DSB-activated axis. Strand invasion of the sister chromatid, which is within the zone of axis-associated inhibition, is thus kinetically impeded; strand invasion of the homolog is unaffected.
Mentions: Our genetic and molecular data indicate that the sister chromatid can be used as efficiently as the homolog in the repair of meiotic DSBs. DSBs that form at hemizygous loci are repaired with the same efficiency and timing as DSBs formed at homozygous loci (Figures 1 and 6A). While these DSBs could, in theory, be repaired by IH gene conversion of the entire region of heterology, such events are relatively rare (Figure 2). We therefore conclude that the majority of DSBs that form at hemizygous loci are repaired by recombination between sister chromatids. Furthermore, the efficient repair of DSBs that form opposite deletions of an entire chromosome arm (Figure 1) indicates that nearby IH interactions are not required for IS recombination to occur. While repair in hemizygous strains occurs exclusively from the sister chromatid, Hunter and colleagues have suggested that multiple templates, including the sister chromatid, are frequently used in the repair of DSBs when both parental homologs are present [11]. JMs containing three and four chromatids form in wild-type cells, and are abundant in strains lacking the Sgs1 helicase [11]. This supports the suggestions that multiple repair templates are often used during meiotic recombination, that recombination is a dynamic process, and that Sgs1 acts to prevent aberrant structures that are formed as a result of these processes [11],[50]. The increased incidence of IS JMs in strains lacking Sgs1 further supports the claim that the sister chromatid is often used for DSB repair during meiosis [11],[12],[49].

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