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Rtt107 phosphorylation promotes localisation to DNA double-stranded breaks (DSBs) and recombinational repair between sister chromatids.

Ullal P, Vilella-Mitjana F, Jarmuz A, Aragón L - PLoS ONE (2011)

Bottom Line: DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break.Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease.Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not.

View Article: PubMed Central - PubMed

Affiliation: Cell Cycle Group, Medical Research Council, Clinical Sciences Centre, Imperial College, London, United Kingdom.

ABSTRACT
Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5-Smc6 complex is important for repair through the SCR pathway.

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Rtt107 phosphorylation is required for its recruitment to the MAT DSB.(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
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pone-0020152-g004: Rtt107 phosphorylation is required for its recruitment to the MAT DSB.(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.

Mentions: Mec1 phosphorylation occurs in Ser/Thr–Gln (SQ/TQ) motifs [23]. A number of SQ/TQ motifs are found scattered within the Rtt107 aminoacid sequence, however a cluster of SQ/TQ motifs at the C-terminal region of Rtt107 have been previously identified as critical for Mec1 phosphorylation [13]. In order to investigate whether phosphorylation in these motifs is required for Rtt107 recruitment to DSBs, we mutated the Ser743, Thr758, Thr773 and Ser806 to alanine (‘A’) residues to generate the Rtt107-AQ allele (Fig. 4A). We expressed the Rtt107-AQ allele tagged with 6 copies of the HA epitope in the C-terminal region under the control of the GAL1-10 promoter in rtt107Δ cells carrying the HO MAT DSB system. The resulting strain was sensitive to a variety of DNA damage agents (data not shown) [13]. We then evaluated the binding of Rtt107-AQ around the HO site. In the absence of a DSB at MAT, we found no Rtt107 binding across the region (Fig. 4A; uncut). After 4 hours of HO induction, we did not find evidence for Rtt107-AQ recruitment to the regions flanking the break (Fig. 4A). We therefore conclude that phosphorylation on the C-terminal SQ/TQ cluster is required for Rtt107 recruitment to the MAT DSB.


Rtt107 phosphorylation promotes localisation to DNA double-stranded breaks (DSBs) and recombinational repair between sister chromatids.

Ullal P, Vilella-Mitjana F, Jarmuz A, Aragón L - PLoS ONE (2011)

Rtt107 phosphorylation is required for its recruitment to the MAT DSB.(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102082&req=5

pone-0020152-g004: Rtt107 phosphorylation is required for its recruitment to the MAT DSB.(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
Mentions: Mec1 phosphorylation occurs in Ser/Thr–Gln (SQ/TQ) motifs [23]. A number of SQ/TQ motifs are found scattered within the Rtt107 aminoacid sequence, however a cluster of SQ/TQ motifs at the C-terminal region of Rtt107 have been previously identified as critical for Mec1 phosphorylation [13]. In order to investigate whether phosphorylation in these motifs is required for Rtt107 recruitment to DSBs, we mutated the Ser743, Thr758, Thr773 and Ser806 to alanine (‘A’) residues to generate the Rtt107-AQ allele (Fig. 4A). We expressed the Rtt107-AQ allele tagged with 6 copies of the HA epitope in the C-terminal region under the control of the GAL1-10 promoter in rtt107Δ cells carrying the HO MAT DSB system. The resulting strain was sensitive to a variety of DNA damage agents (data not shown) [13]. We then evaluated the binding of Rtt107-AQ around the HO site. In the absence of a DSB at MAT, we found no Rtt107 binding across the region (Fig. 4A; uncut). After 4 hours of HO induction, we did not find evidence for Rtt107-AQ recruitment to the regions flanking the break (Fig. 4A). We therefore conclude that phosphorylation on the C-terminal SQ/TQ cluster is required for Rtt107 recruitment to the MAT DSB.

Bottom Line: DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break.Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease.Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not.

View Article: PubMed Central - PubMed

Affiliation: Cell Cycle Group, Medical Research Council, Clinical Sciences Centre, Imperial College, London, United Kingdom.

ABSTRACT
Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5-Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5-Smc6 complex is important for repair through the SCR pathway.

Show MeSH
Related in: MedlinePlus