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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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Slx4 and Rtt101 are not required for Rtt107 recruitment to the MAT DSB.(A) Slx4 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and slx4Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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. (B) Rtt101 deletion does not prevent Rtt107 recruitment to HO-induced DSBs. A DSB was induced at the MAT locus in wildtype (CCG6983) and rtt101Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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-g005: Slx4 and Rtt101 are not required for Rtt107 recruitment to the MAT DSB.(A) Slx4 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and slx4Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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. (B) Rtt101 deletion does not prevent Rtt107 recruitment to HO-induced DSBs. A DSB was induced at the MAT locus in wildtype (CCG6983) and rtt101Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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: The N-terminal region of Rtt107 contains four tandem BRCT domains, which are characteristic of proteins involved in cell cycle checkpoint functions related to DNA damage [26]. These domains have been shown to interact with a number of proteins, including the Slx4/Slx1 nuclease complex [14]. In addition, Rtt107 is recruited to stalled replication forks via interaction with the cullin Rtt101 protein [17]. We therefore wished to address whether these interactions are important for the observed recruitment of Rtt107 to DSBs. First, we investigated whether Rtt107 is recruited to DSBs in the absence of Slx4. Slx4Δ largely prevents Rtt107 SQ/TQ phosphorylation [14], a requirement for Rtt107 DSB recruitment (Fig. 4). Surprisingly, we found no significant defects in Rtt107 recruitment to MAT DSBs in the absence of Slx4 (Fig. 5A). Next we tested whether the cullin Rtt101 is required for the localization of Rtt107 to breaks. We found Rtt107 recruitment to HO-induced DSBs to be normal in rtt101Δ cells (Fig. 5B). Therefore, we conclude that while the Slx4/Slx1 and Rtt101 interactions might be important for Rtt107 functions at damaged forks [14], [17] they are not essential for its recruitment to DSBs induced by HO.


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)

Slx4 and Rtt101 are not required for Rtt107 recruitment to the MAT DSB.(A) Slx4 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and slx4Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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. (B) Rtt101 deletion does not prevent Rtt107 recruitment to HO-induced DSBs. A DSB was induced at the MAT locus in wildtype (CCG6983) and rtt101Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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-g005: Slx4 and Rtt101 are not required for Rtt107 recruitment to the MAT DSB.(A) Slx4 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and slx4Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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. (B) Rtt101 deletion does not prevent Rtt107 recruitment to HO-induced DSBs. A DSB was induced at the MAT locus in wildtype (CCG6983) and rtt101Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 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: The N-terminal region of Rtt107 contains four tandem BRCT domains, which are characteristic of proteins involved in cell cycle checkpoint functions related to DNA damage [26]. These domains have been shown to interact with a number of proteins, including the Slx4/Slx1 nuclease complex [14]. In addition, Rtt107 is recruited to stalled replication forks via interaction with the cullin Rtt101 protein [17]. We therefore wished to address whether these interactions are important for the observed recruitment of Rtt107 to DSBs. First, we investigated whether Rtt107 is recruited to DSBs in the absence of Slx4. Slx4Δ largely prevents Rtt107 SQ/TQ phosphorylation [14], a requirement for Rtt107 DSB recruitment (Fig. 4). Surprisingly, we found no significant defects in Rtt107 recruitment to MAT DSBs in the absence of Slx4 (Fig. 5A). Next we tested whether the cullin Rtt101 is required for the localization of Rtt107 to breaks. We found Rtt107 recruitment to HO-induced DSBs to be normal in rtt101Δ cells (Fig. 5B). Therefore, we conclude that while the Slx4/Slx1 and Rtt101 interactions might be important for Rtt107 functions at damaged forks [14], [17] they are not essential for its recruitment to DSBs induced by HO.

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