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TLK1B promotes repair of DSBs via its interaction with Rad9 and Asf1.

Canfield C, Rains J, De Benedetti A - BMC Mol. Biol. (2009)

Bottom Line: However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9.From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, 71130, USA. callae.canfield@gmail.com

ABSTRACT

Background: The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.

Results: Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B.

Conclusions: TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.

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Model for the activity of TLK1/1B in translesion repair. Rad9 is known to be involved in translesion repair synthesis [34]. TLK1B helps modulating the activity and assembly of the 9-1-1 complex and promoting repair-coupled chromatin remodeling which depends on Asf1. Integration of the two activities is that TLK1/1B is first recruited to a DSB in a complex with 9-1-1 and the RFC-Rad17 clamp loader, to which Asf1 also binds [47]. At this point, TLK1/1B exchanges with Asf1 to promote nucleosomes eviction and access of the repair machinery to unencumbered DNA [11]. Faster repair can thus take place and is also followed by more rapid reassembly of chromatin, which is believed to be the real signal for resumption of the cell cycle [39]. We suggest that in DSB repair, Rad9 activity on ends-processing has an even more important role in repair [51].
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Figure 7: Model for the activity of TLK1/1B in translesion repair. Rad9 is known to be involved in translesion repair synthesis [34]. TLK1B helps modulating the activity and assembly of the 9-1-1 complex and promoting repair-coupled chromatin remodeling which depends on Asf1. Integration of the two activities is that TLK1/1B is first recruited to a DSB in a complex with 9-1-1 and the RFC-Rad17 clamp loader, to which Asf1 also binds [47]. At this point, TLK1/1B exchanges with Asf1 to promote nucleosomes eviction and access of the repair machinery to unencumbered DNA [11]. Faster repair can thus take place and is also followed by more rapid reassembly of chromatin, which is believed to be the real signal for resumption of the cell cycle [39]. We suggest that in DSB repair, Rad9 activity on ends-processing has an even more important role in repair [51].

Mentions: Asf1 is known to interact with RFC (subunits 2-5) tethered to PCNA, and it is recruited to the replication forks [47] We propose that after DNA damage, Asf1 is similarly recruited to the lesions to prepare for repair. In this capacity, Asf1 may be instead recruited by the Rad17-RFC clamp-loader, just as Rad9 is, in association with TLK1/1B. After dissociation from RFC [47], the recruited Asf1 is positioned to disrupt the H3/H4 tetramer resulting in nucleosome eviction. As repair progresses, newly synthesized TLK1B induced by DNA damage, or TLK1B already overexpressed, leads to dissociation of the Asf1/H3/H4 heterotrimer thus promoting the formation of the core tetramer [44]. The H3/H4 tetramer may then be redeposited onto repaired DNA by the HIR complex [48] without necessarily Asf1 participation (Fig. 7). Many details remain to be filled-in - for instance the role that ATM plays in modulating the two separate activities of TLK1 (kinase and chaperone). We recently showed that the association of TLK1B with Asf1 is regulated by its phosphorylation [44]. Thus, a possible outcome for the role of ATM-mediated inhibition of TLK1/1B is that the resulting reduction of Asf1 phosphorylation would lead to a more stable association of TLK1/1B-Asf1, instead of a kinetic association between the two proteins involving the ratio of unphosphorylated and phosphorylated Asf1. This could also lead to dissociation of the Asf1/H3/H4 heterotrimer. Another question is how the Rad9-mediated checkpoint activation of ATM and ATR may affect the entire pathway and its own association with TLK1/1B and Rad17. In addition, since TLK1 kinase activity is rapidly inhibited after DSBs, this could result in accumulation of dephosphorylated (S328) Rad9. After TLK1/1B kinase activity is restored after repair, Rad9 may then be re-phosphorylated, which could be an important mark for release of the clamp complex and signaling completion of repair and resumption of the cell cycle.


TLK1B promotes repair of DSBs via its interaction with Rad9 and Asf1.

Canfield C, Rains J, De Benedetti A - BMC Mol. Biol. (2009)

Model for the activity of TLK1/1B in translesion repair. Rad9 is known to be involved in translesion repair synthesis [34]. TLK1B helps modulating the activity and assembly of the 9-1-1 complex and promoting repair-coupled chromatin remodeling which depends on Asf1. Integration of the two activities is that TLK1/1B is first recruited to a DSB in a complex with 9-1-1 and the RFC-Rad17 clamp loader, to which Asf1 also binds [47]. At this point, TLK1/1B exchanges with Asf1 to promote nucleosomes eviction and access of the repair machinery to unencumbered DNA [11]. Faster repair can thus take place and is also followed by more rapid reassembly of chromatin, which is believed to be the real signal for resumption of the cell cycle [39]. We suggest that in DSB repair, Rad9 activity on ends-processing has an even more important role in repair [51].
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Related In: Results  -  Collection

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Figure 7: Model for the activity of TLK1/1B in translesion repair. Rad9 is known to be involved in translesion repair synthesis [34]. TLK1B helps modulating the activity and assembly of the 9-1-1 complex and promoting repair-coupled chromatin remodeling which depends on Asf1. Integration of the two activities is that TLK1/1B is first recruited to a DSB in a complex with 9-1-1 and the RFC-Rad17 clamp loader, to which Asf1 also binds [47]. At this point, TLK1/1B exchanges with Asf1 to promote nucleosomes eviction and access of the repair machinery to unencumbered DNA [11]. Faster repair can thus take place and is also followed by more rapid reassembly of chromatin, which is believed to be the real signal for resumption of the cell cycle [39]. We suggest that in DSB repair, Rad9 activity on ends-processing has an even more important role in repair [51].
Mentions: Asf1 is known to interact with RFC (subunits 2-5) tethered to PCNA, and it is recruited to the replication forks [47] We propose that after DNA damage, Asf1 is similarly recruited to the lesions to prepare for repair. In this capacity, Asf1 may be instead recruited by the Rad17-RFC clamp-loader, just as Rad9 is, in association with TLK1/1B. After dissociation from RFC [47], the recruited Asf1 is positioned to disrupt the H3/H4 tetramer resulting in nucleosome eviction. As repair progresses, newly synthesized TLK1B induced by DNA damage, or TLK1B already overexpressed, leads to dissociation of the Asf1/H3/H4 heterotrimer thus promoting the formation of the core tetramer [44]. The H3/H4 tetramer may then be redeposited onto repaired DNA by the HIR complex [48] without necessarily Asf1 participation (Fig. 7). Many details remain to be filled-in - for instance the role that ATM plays in modulating the two separate activities of TLK1 (kinase and chaperone). We recently showed that the association of TLK1B with Asf1 is regulated by its phosphorylation [44]. Thus, a possible outcome for the role of ATM-mediated inhibition of TLK1/1B is that the resulting reduction of Asf1 phosphorylation would lead to a more stable association of TLK1/1B-Asf1, instead of a kinetic association between the two proteins involving the ratio of unphosphorylated and phosphorylated Asf1. This could also lead to dissociation of the Asf1/H3/H4 heterotrimer. Another question is how the Rad9-mediated checkpoint activation of ATM and ATR may affect the entire pathway and its own association with TLK1/1B and Rad17. In addition, since TLK1 kinase activity is rapidly inhibited after DSBs, this could result in accumulation of dephosphorylated (S328) Rad9. After TLK1/1B kinase activity is restored after repair, Rad9 may then be re-phosphorylated, which could be an important mark for release of the clamp complex and signaling completion of repair and resumption of the cell cycle.

Bottom Line: However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9.From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and the Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, 71130, USA. callae.canfield@gmail.com

ABSTRACT

Background: The Tousled-like kinases are involved in chromatin assembly, DNA repair, transcription, and chromosome segregation. Previous evidence indicated that TLK1B can promote repair of plasmids with cohesive ends in vitro, but it was inferred that the mechanism was indirect and via chromatin assembly, mediated by its interaction with the chromatin assembly factor Asf1. We recently identified Rad9 as a substrate of TLK1B, and we presented evidence that the TLK1B-Rad9 interaction plays some role in DSB repair. Hence the relative contribution of Asf1 and Rad9 to the protective effect of TLK1B in DSBs repair is not known. Using an adeno-HO-mediated cleavage system in MM3MG cells, we previously showed that overexpression of either TLK1B or a kinase-dead protein (KD) promoted repair and the assembly of Rad9 in proximity of the DSB at early time points post-infection. This established that it is a chaperone activity of TLK1B and not directly the kinase activity that promotes recruitment of 9-1-1 to the DSB. However, the phosphorylation of Rad9(S328) by TLK1B appeared important for mediating a cell cycle checkpoint, and thus, this phosphorylation of Rad9 may have other effects on 9-1-1 functionality.

Results: Here we present direct evidence that TLK1B can promote repair of linearized plasmids with incompatible ends that require processing prior to ligation. Immunodepletion of Rad9 indicated that Rad9 was important for processing the ends preceding ligation, suggesting that the interaction of TLK1B with Rad9 is a key mediator for this type of repair. Ligation of incompatible ends also required DNA-PK, as addition of wortmannin or immunodepletion of Ku70 abrogated ligation. Depletion of Ku70 prevented the ligation of the plasmid but did not affect stimulation of the fill-in of the ends by added TLK1B, which was attributed to Rad9. From experiments with the HO-cleavage system, we now show that Rad17, a subunit of the "clamp loader", associates normally with the DSB in KD-overexpressing cells. However, the subsequent release of Rad17 and Rad9 upon repair of the DSB was significantly slower in these cells compared to controls or cells expressing wt-TLK1B.

Conclusions: TLKs play important roles in DNA repair, not only by modulation of chromatin assembly via Asf1, but also by a more direct function in processing the ends of a DSB via interaction with Rad9. Inhibition of Rad9 phosphorylation in KD-overexpressing cells may have consequences in signaling completion of the repair and cell cycle re-entry, and could explain a loss of viability from DSBs in these cells.

Show MeSH