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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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Ligation and supercoiling does not depend on Rad9 when the ends are already blunt. The plasmid cut with EcoRI and EcoRV was pre-labeled and filled-in with [α-32P]dATP and dTTP. Rad9-depeleted extract was used for these repair reactions, and the effect of the addition of TLK1B or the KD was also shown.
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Figure 3: Ligation and supercoiling does not depend on Rad9 when the ends are already blunt. The plasmid cut with EcoRI and EcoRV was pre-labeled and filled-in with [α-32P]dATP and dTTP. Rad9-depeleted extract was used for these repair reactions, and the effect of the addition of TLK1B or the KD was also shown.

Mentions: To establish if the main effect of Rad9 was to polish the ends to create conditions suitable for blunt-end ligation, the plasmid was filled-in with Klenow polymerase and [α32P]dATP and dTTP, and incubated with extract depleted of Rad9. As seen in Fig. 3, the extract was in this case capable of promoting ligation/supercoiling, confirming that the generation of flush ends is really the limiting step for repair of incompatible ends in these reactions. The addition of TLK1B or the kinase-inactive mutant (KD) stimulated the formation of the more highly supercoiled forms. This effect can clearly be attributed to the effect of TLK1B on Asf1, and is consistent with our previous report that it is the chaperone function of TLK1B, not its kinase activity, that is responsible for stimulation of nucleosome formation [11]. Furthermore, TLK1B can counteract Asf1's inhibition of H3/H4 tetramerization in the absence of ATP [44]


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

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

Ligation and supercoiling does not depend on Rad9 when the ends are already blunt. The plasmid cut with EcoRI and EcoRV was pre-labeled and filled-in with [α-32P]dATP and dTTP. Rad9-depeleted extract was used for these repair reactions, and the effect of the addition of TLK1B or the KD was also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803485&req=5

Figure 3: Ligation and supercoiling does not depend on Rad9 when the ends are already blunt. The plasmid cut with EcoRI and EcoRV was pre-labeled and filled-in with [α-32P]dATP and dTTP. Rad9-depeleted extract was used for these repair reactions, and the effect of the addition of TLK1B or the KD was also shown.
Mentions: To establish if the main effect of Rad9 was to polish the ends to create conditions suitable for blunt-end ligation, the plasmid was filled-in with Klenow polymerase and [α32P]dATP and dTTP, and incubated with extract depleted of Rad9. As seen in Fig. 3, the extract was in this case capable of promoting ligation/supercoiling, confirming that the generation of flush ends is really the limiting step for repair of incompatible ends in these reactions. The addition of TLK1B or the kinase-inactive mutant (KD) stimulated the formation of the more highly supercoiled forms. This effect can clearly be attributed to the effect of TLK1B on Asf1, and is consistent with our previous report that it is the chaperone function of TLK1B, not its kinase activity, that is responsible for stimulation of nucleosome formation [11]. Furthermore, TLK1B can counteract Asf1's inhibition of H3/H4 tetramerization in the absence of ATP [44]

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