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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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Repair of linearized palsmids and dependence on TLK1, Rad9, and Ku70. A) Western blots of extracts depleted of TLK1, Rad9, and Ku70. B) Immunodepletion of TLK1 and add-back. The effect of immunodepleting the endogenous TLK1 in labeling of the ends and ligation/supercoiling is shown. In the middle lanes, recombinant TLK1B was added back to the immunodepleted extract in an amount comparable to the endogenous TLK1 (see panel A). Note that this gel was exposed for longer than that in Fig. 1C to reveal the repaired forms more clearly. Quantitation of the autorad in pixels is shown below each lane. C)  Plasmid repair dependence on Rad9. Where indicated, the extract was immunodepleted of Rad9. In the left panel, we monitored repair of plasmid linearized with EcoRI alone (cohesive ends repair). In the middle panel, we monitored labeling and religation/supercoiling of plasmid cut with EcoRI/EcoRV, and its dependence on Rad9. In the right panel, Rad9 was added back. The position of linear and two supercoiled forms of the plasmids are indicated. D) Plasmid repair dependence on Ku70 and DNA-PK. The plasmid was in this case pre-labeled with Kleonw polymerase and [α-32P]dATP. Where indicated, the extract was immunodepleted of Ku (left panel), or the reaction was carried out in presence of 1 μM wortmainnin (right panel). Where indicated, we added TLK1B.
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Figure 2: Repair of linearized palsmids and dependence on TLK1, Rad9, and Ku70. A) Western blots of extracts depleted of TLK1, Rad9, and Ku70. B) Immunodepletion of TLK1 and add-back. The effect of immunodepleting the endogenous TLK1 in labeling of the ends and ligation/supercoiling is shown. In the middle lanes, recombinant TLK1B was added back to the immunodepleted extract in an amount comparable to the endogenous TLK1 (see panel A). Note that this gel was exposed for longer than that in Fig. 1C to reveal the repaired forms more clearly. Quantitation of the autorad in pixels is shown below each lane. C) Plasmid repair dependence on Rad9. Where indicated, the extract was immunodepleted of Rad9. In the left panel, we monitored repair of plasmid linearized with EcoRI alone (cohesive ends repair). In the middle panel, we monitored labeling and religation/supercoiling of plasmid cut with EcoRI/EcoRV, and its dependence on Rad9. In the right panel, Rad9 was added back. The position of linear and two supercoiled forms of the plasmids are indicated. D) Plasmid repair dependence on Ku70 and DNA-PK. The plasmid was in this case pre-labeled with Kleonw polymerase and [α-32P]dATP. Where indicated, the extract was immunodepleted of Ku (left panel), or the reaction was carried out in presence of 1 μM wortmainnin (right panel). Where indicated, we added TLK1B.

Mentions: So far, it was unclear if the effect of TLK1B in repair was due to a more rapid fill-in (via Rad9) that may be a prerequisite for blunt-ends ligation or whether the effect was instead mediated by Asf1 by plasmid compaction (or due to both). To test this, the fill-in reaction and repair was studied in extract depleted of Ku70 (Fig. 1D and see also Fig. 2), as DNA-PK/Ku is required for ligation of such ends (reviewed in [41]. Hence, the fill-in reaction could be studied separately from the ligation/supercoiling effect. Under these condition, TLK1B stimulated the labeling of the ends (fill-in), whereas almost no ligation, inferred by supercoiling, could be seen (Fig. 1D).


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

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

Repair of linearized palsmids and dependence on TLK1, Rad9, and Ku70. A) Western blots of extracts depleted of TLK1, Rad9, and Ku70. B) Immunodepletion of TLK1 and add-back. The effect of immunodepleting the endogenous TLK1 in labeling of the ends and ligation/supercoiling is shown. In the middle lanes, recombinant TLK1B was added back to the immunodepleted extract in an amount comparable to the endogenous TLK1 (see panel A). Note that this gel was exposed for longer than that in Fig. 1C to reveal the repaired forms more clearly. Quantitation of the autorad in pixels is shown below each lane. C)  Plasmid repair dependence on Rad9. Where indicated, the extract was immunodepleted of Rad9. In the left panel, we monitored repair of plasmid linearized with EcoRI alone (cohesive ends repair). In the middle panel, we monitored labeling and religation/supercoiling of plasmid cut with EcoRI/EcoRV, and its dependence on Rad9. In the right panel, Rad9 was added back. The position of linear and two supercoiled forms of the plasmids are indicated. D) Plasmid repair dependence on Ku70 and DNA-PK. The plasmid was in this case pre-labeled with Kleonw polymerase and [α-32P]dATP. Where indicated, the extract was immunodepleted of Ku (left panel), or the reaction was carried out in presence of 1 μM wortmainnin (right panel). Where indicated, we added TLK1B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Repair of linearized palsmids and dependence on TLK1, Rad9, and Ku70. A) Western blots of extracts depleted of TLK1, Rad9, and Ku70. B) Immunodepletion of TLK1 and add-back. The effect of immunodepleting the endogenous TLK1 in labeling of the ends and ligation/supercoiling is shown. In the middle lanes, recombinant TLK1B was added back to the immunodepleted extract in an amount comparable to the endogenous TLK1 (see panel A). Note that this gel was exposed for longer than that in Fig. 1C to reveal the repaired forms more clearly. Quantitation of the autorad in pixels is shown below each lane. C) Plasmid repair dependence on Rad9. Where indicated, the extract was immunodepleted of Rad9. In the left panel, we monitored repair of plasmid linearized with EcoRI alone (cohesive ends repair). In the middle panel, we monitored labeling and religation/supercoiling of plasmid cut with EcoRI/EcoRV, and its dependence on Rad9. In the right panel, Rad9 was added back. The position of linear and two supercoiled forms of the plasmids are indicated. D) Plasmid repair dependence on Ku70 and DNA-PK. The plasmid was in this case pre-labeled with Kleonw polymerase and [α-32P]dATP. Where indicated, the extract was immunodepleted of Ku (left panel), or the reaction was carried out in presence of 1 μM wortmainnin (right panel). Where indicated, we added TLK1B.
Mentions: So far, it was unclear if the effect of TLK1B in repair was due to a more rapid fill-in (via Rad9) that may be a prerequisite for blunt-ends ligation or whether the effect was instead mediated by Asf1 by plasmid compaction (or due to both). To test this, the fill-in reaction and repair was studied in extract depleted of Ku70 (Fig. 1D and see also Fig. 2), as DNA-PK/Ku is required for ligation of such ends (reviewed in [41]. Hence, the fill-in reaction could be studied separately from the ligation/supercoiling effect. Under these condition, TLK1B stimulated the labeling of the ends (fill-in), whereas almost no ligation, inferred by supercoiling, could be seen (Fig. 1D).

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
Related in: MedlinePlus