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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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Related in: MedlinePlus

Similar timing of IS and IH DSB repair.(A) Structure of DSB hotspots in a 3.5-kb his4::URA3-arg4 insert [29] and in the YCR047c–YCR048w intergenic region [82]. White boxes indicate URA3-arg4 insert genes; grey boxes indicate other genes on chromosome III; horizontal bars indicate sequences used for probes [8],[29]; vertical lines indicate restriction sites used to detect DSBs (short lines) and JM intermediates (long lines). (B) Strains used. (i) Homozygous control—his4::URA3-arg4 and YCR047c–YCR048w are present on both homologs. (ii) Hemizygous at his4::URA3-arg4—the 3.5-kb his4::URA3-arg4 insert is present on only one homolog. (iii) Left arm deletion—the his4::URA3-arg4 insert is present on one homolog, opposite a 90-kb deletion on the other homolog. (iv) Hemizygous at YCR047c—4 kb of DNA between YCR046c and YCR051w is deleted from one homolog. (C) Southern blots showing detection of DSBs in wild-type and rad50S strains hemizygous for his4::URA3-arg4 at either the his4::URA3-arg4 site (left) or the YCR047c homozygous control site (right). P, parental band; DSB, DSB band. (D) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains are (right-hand y-axis). (E) DSB life span, calculated using 7-h rad50S cumulative DSB levels, as described previously [81]. Underlined life spans are for DSBs that must be repaired by IS recombination. Strains in (C–E) are (for RAD50 and rad50S, respectively): homozygous, MJL3201 and MJL3198; hemizygous at his4::URA3-arg4, MJL3250 and MJL3338; left arm deletion, MJL3227 and MJL3233; and hemizygous at YCR047c, MJL3399 and MJL3408.
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pbio-1000520-g001: Similar timing of IS and IH DSB repair.(A) Structure of DSB hotspots in a 3.5-kb his4::URA3-arg4 insert [29] and in the YCR047c–YCR048w intergenic region [82]. White boxes indicate URA3-arg4 insert genes; grey boxes indicate other genes on chromosome III; horizontal bars indicate sequences used for probes [8],[29]; vertical lines indicate restriction sites used to detect DSBs (short lines) and JM intermediates (long lines). (B) Strains used. (i) Homozygous control—his4::URA3-arg4 and YCR047c–YCR048w are present on both homologs. (ii) Hemizygous at his4::URA3-arg4—the 3.5-kb his4::URA3-arg4 insert is present on only one homolog. (iii) Left arm deletion—the his4::URA3-arg4 insert is present on one homolog, opposite a 90-kb deletion on the other homolog. (iv) Hemizygous at YCR047c—4 kb of DNA between YCR046c and YCR051w is deleted from one homolog. (C) Southern blots showing detection of DSBs in wild-type and rad50S strains hemizygous for his4::URA3-arg4 at either the his4::URA3-arg4 site (left) or the YCR047c homozygous control site (right). P, parental band; DSB, DSB band. (D) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains are (right-hand y-axis). (E) DSB life span, calculated using 7-h rad50S cumulative DSB levels, as described previously [81]. Underlined life spans are for DSBs that must be repaired by IS recombination. Strains in (C–E) are (for RAD50 and rad50S, respectively): homozygous, MJL3201 and MJL3198; hemizygous at his4::URA3-arg4, MJL3250 and MJL3338; left arm deletion, MJL3227 and MJL3233; and hemizygous at YCR047c, MJL3399 and MJL3408.

Mentions: We examined DSBs at two hotspots on chromosome III: within a 3.5-kb recombination reporter construct containing URA3 and ARG4 sequences inserted at HIS4 (his4::URA3-arg4; [29]) and in the YCR047c promoter (Figure 1A). Both DSB hotspots were examined in a hemizygous configuration, where the hotspot was present on one copy of chromosome III and a small deletion was present on the other homolog. This eliminates the possibility of repair of the hotspot DSB by IH recombination (Figure 1B), but preserves normal homolog alignment, synapsis, and IH recombination in the genome as a whole. We also examined a strain hemizygous for a deletion that removes most of the chromosome III left arm, including sequences for about 45 kb to either side of the his4::URA3-arg4 insertion site (Figure 1B). In most experiments, DSB dynamics were examined in the same strain at a hemizygous site and at a homozygous control site, to control for culture-to-culture variation in meiotic progression and the fraction of cells undergoing meiosis.


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)

Similar timing of IS and IH DSB repair.(A) Structure of DSB hotspots in a 3.5-kb his4::URA3-arg4 insert [29] and in the YCR047c–YCR048w intergenic region [82]. White boxes indicate URA3-arg4 insert genes; grey boxes indicate other genes on chromosome III; horizontal bars indicate sequences used for probes [8],[29]; vertical lines indicate restriction sites used to detect DSBs (short lines) and JM intermediates (long lines). (B) Strains used. (i) Homozygous control—his4::URA3-arg4 and YCR047c–YCR048w are present on both homologs. (ii) Hemizygous at his4::URA3-arg4—the 3.5-kb his4::URA3-arg4 insert is present on only one homolog. (iii) Left arm deletion—the his4::URA3-arg4 insert is present on one homolog, opposite a 90-kb deletion on the other homolog. (iv) Hemizygous at YCR047c—4 kb of DNA between YCR046c and YCR051w is deleted from one homolog. (C) Southern blots showing detection of DSBs in wild-type and rad50S strains hemizygous for his4::URA3-arg4 at either the his4::URA3-arg4 site (left) or the YCR047c homozygous control site (right). P, parental band; DSB, DSB band. (D) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains are (right-hand y-axis). (E) DSB life span, calculated using 7-h rad50S cumulative DSB levels, as described previously [81]. Underlined life spans are for DSBs that must be repaired by IS recombination. Strains in (C–E) are (for RAD50 and rad50S, respectively): homozygous, MJL3201 and MJL3198; hemizygous at his4::URA3-arg4, MJL3250 and MJL3338; left arm deletion, MJL3227 and MJL3233; and hemizygous at YCR047c, MJL3399 and MJL3408.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1000520-g001: Similar timing of IS and IH DSB repair.(A) Structure of DSB hotspots in a 3.5-kb his4::URA3-arg4 insert [29] and in the YCR047c–YCR048w intergenic region [82]. White boxes indicate URA3-arg4 insert genes; grey boxes indicate other genes on chromosome III; horizontal bars indicate sequences used for probes [8],[29]; vertical lines indicate restriction sites used to detect DSBs (short lines) and JM intermediates (long lines). (B) Strains used. (i) Homozygous control—his4::URA3-arg4 and YCR047c–YCR048w are present on both homologs. (ii) Hemizygous at his4::URA3-arg4—the 3.5-kb his4::URA3-arg4 insert is present on only one homolog. (iii) Left arm deletion—the his4::URA3-arg4 insert is present on one homolog, opposite a 90-kb deletion on the other homolog. (iv) Hemizygous at YCR047c—4 kb of DNA between YCR046c and YCR051w is deleted from one homolog. (C) Southern blots showing detection of DSBs in wild-type and rad50S strains hemizygous for his4::URA3-arg4 at either the his4::URA3-arg4 site (left) or the YCR047c homozygous control site (right). P, parental band; DSB, DSB band. (D) DSB frequencies (3–4 independent experiments, error bars indicate SEM), quantified as percent of total lane signal. Symbols connected by lines are noncumulative DSB frequencies from RAD50 strains (left-hand y-axis); unconnected symbols at 7 h are cumulative DSB frequencies from rad50S strains are (right-hand y-axis). (E) DSB life span, calculated using 7-h rad50S cumulative DSB levels, as described previously [81]. Underlined life spans are for DSBs that must be repaired by IS recombination. Strains in (C–E) are (for RAD50 and rad50S, respectively): homozygous, MJL3201 and MJL3198; hemizygous at his4::URA3-arg4, MJL3250 and MJL3338; left arm deletion, MJL3227 and MJL3233; and hemizygous at YCR047c, MJL3399 and MJL3408.
Mentions: We examined DSBs at two hotspots on chromosome III: within a 3.5-kb recombination reporter construct containing URA3 and ARG4 sequences inserted at HIS4 (his4::URA3-arg4; [29]) and in the YCR047c promoter (Figure 1A). Both DSB hotspots were examined in a hemizygous configuration, where the hotspot was present on one copy of chromosome III and a small deletion was present on the other homolog. This eliminates the possibility of repair of the hotspot DSB by IH recombination (Figure 1B), but preserves normal homolog alignment, synapsis, and IH recombination in the genome as a whole. We also examined a strain hemizygous for a deletion that removes most of the chromosome III left arm, including sequences for about 45 kb to either side of the his4::URA3-arg4 insertion site (Figure 1B). In most experiments, DSB dynamics were examined in the same strain at a hemizygous site and at a homozygous control site, to control for culture-to-culture variation in meiotic progression and the fraction of cells undergoing meiosis.

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