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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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Genomic HO-mediated cleavage and repair. A) Diagram of the Mat cassette integrated at chromosome 14. These cell lines were described in [11]. B) Time course Post Infection (PI) with adeno-HO. Presence of the DSB was followed by PCR, probing for a ~500 bp product generated with primers T7 and Puro2 flanking the HO-site. MM3MG cells and cells overexpressing TLK1B-KD (kinase-dead) were compared for kinetics of DSB repair. For greater sensitivity, the PCR reaction included [α-32P]dATP, and 23 cycles were employed. FABP1 (fat binding protein 1) is a control product from a single copy gene. C) Western blot of E3::HO. This is shown during a time course of Adeno-HO infection.
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Figure 4: Genomic HO-mediated cleavage and repair. A) Diagram of the Mat cassette integrated at chromosome 14. These cell lines were described in [11]. B) Time course Post Infection (PI) with adeno-HO. Presence of the DSB was followed by PCR, probing for a ~500 bp product generated with primers T7 and Puro2 flanking the HO-site. MM3MG cells and cells overexpressing TLK1B-KD (kinase-dead) were compared for kinetics of DSB repair. For greater sensitivity, the PCR reaction included [α-32P]dATP, and 23 cycles were employed. FABP1 (fat binding protein 1) is a control product from a single copy gene. C) Western blot of E3::HO. This is shown during a time course of Adeno-HO infection.

Mentions: We have recently described a system in MM3MG cells that allows to study the repair of a single genomic DSB using the yeast HO-nuclease expressed from adenovirus [11]; a diagram of the HO target cassette shown in Fig. 4A. We have repeated here a time course of HO cleavage to reintroduce the system. During a time course of infection, the HO endonuclease generated a single DSB at the HO-site, which could be monitored as a loss of a PCR product from genomic DNA with primers flanking the HO site (amplicon T7/Puro2). At an MOI of 300, the cleavage is complete at 3 h post-infection (PI), after which, repair occurs mostly by high-fidelity religation (Fig. 4B). To control for input DNA, a different amplicon was generated from the single-copy FABP1 gene on chromosome 6. This time-course of repair coincides with the pattern of expression of HO endonuclease, which peaks after 1 h and is then largely degraded by 3 h PI (Fig. 4C), in both control or KD-expressing cells. We have also reported that in the isogenic MM3MG cells overexpressing TLK1B or the KD protein, the progress of DSB repair was somewhat faster [11]. This was reproduced here (compare the 6 h time-point), and it confirmed that the kinase activity of TLK1B is not required for all the functions of this protein in DSB repair. Of course, the kinase is absolutely required for the phosphorylation of Rad9 at S328 and that of Asf1. We should also mention that, despite the fact that the KD-expressing cells can repair the HO-mediated cleavage with comparable or even faster kinetics than the control cells, they are nonetheless very sensitive to IR-mediated DSBs [4]. This may be related to the incapacity to phosphorylate Rad9 [11]. We have reported that cells overexpressing TLK1B or the KD recruited Rad9 in proximity of the DSB to much greater level than control cells, consistent with the specific interaction of TLK1B with Rad9. We postulated that the TLK1B-Rad9 interaction promotes and/or stabilizes their association with the DSB. Since it is the Rad17-RFC clamp-loader that locks the 9-1-1 complex onto damaged DNA [25], we wanted to test if the occupancy of Rad17 in proximity of the DSB was altered in KD-overexpressing cells. We reasoned that the recruitment of Rad17 to the DSB should not be affected by the KD, as there is no direct interaction of Rad17 with TLK1B, and further the binding of the clamp loader to DSBs precedes 9-1-1 assembly. On the other hand, it seemed possible that the phosphorylation of Rad9 cells could somehow affect the association or stability of the Rad17-RFC/9-1-1 complex with the DSB.


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

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

Genomic HO-mediated cleavage and repair. A) Diagram of the Mat cassette integrated at chromosome 14. These cell lines were described in [11]. B) Time course Post Infection (PI) with adeno-HO. Presence of the DSB was followed by PCR, probing for a ~500 bp product generated with primers T7 and Puro2 flanking the HO-site. MM3MG cells and cells overexpressing TLK1B-KD (kinase-dead) were compared for kinetics of DSB repair. For greater sensitivity, the PCR reaction included [α-32P]dATP, and 23 cycles were employed. FABP1 (fat binding protein 1) is a control product from a single copy gene. C) Western blot of E3::HO. This is shown during a time course of Adeno-HO infection.
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Related In: Results  -  Collection

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Figure 4: Genomic HO-mediated cleavage and repair. A) Diagram of the Mat cassette integrated at chromosome 14. These cell lines were described in [11]. B) Time course Post Infection (PI) with adeno-HO. Presence of the DSB was followed by PCR, probing for a ~500 bp product generated with primers T7 and Puro2 flanking the HO-site. MM3MG cells and cells overexpressing TLK1B-KD (kinase-dead) were compared for kinetics of DSB repair. For greater sensitivity, the PCR reaction included [α-32P]dATP, and 23 cycles were employed. FABP1 (fat binding protein 1) is a control product from a single copy gene. C) Western blot of E3::HO. This is shown during a time course of Adeno-HO infection.
Mentions: We have recently described a system in MM3MG cells that allows to study the repair of a single genomic DSB using the yeast HO-nuclease expressed from adenovirus [11]; a diagram of the HO target cassette shown in Fig. 4A. We have repeated here a time course of HO cleavage to reintroduce the system. During a time course of infection, the HO endonuclease generated a single DSB at the HO-site, which could be monitored as a loss of a PCR product from genomic DNA with primers flanking the HO site (amplicon T7/Puro2). At an MOI of 300, the cleavage is complete at 3 h post-infection (PI), after which, repair occurs mostly by high-fidelity religation (Fig. 4B). To control for input DNA, a different amplicon was generated from the single-copy FABP1 gene on chromosome 6. This time-course of repair coincides with the pattern of expression of HO endonuclease, which peaks after 1 h and is then largely degraded by 3 h PI (Fig. 4C), in both control or KD-expressing cells. We have also reported that in the isogenic MM3MG cells overexpressing TLK1B or the KD protein, the progress of DSB repair was somewhat faster [11]. This was reproduced here (compare the 6 h time-point), and it confirmed that the kinase activity of TLK1B is not required for all the functions of this protein in DSB repair. Of course, the kinase is absolutely required for the phosphorylation of Rad9 at S328 and that of Asf1. We should also mention that, despite the fact that the KD-expressing cells can repair the HO-mediated cleavage with comparable or even faster kinetics than the control cells, they are nonetheless very sensitive to IR-mediated DSBs [4]. This may be related to the incapacity to phosphorylate Rad9 [11]. We have reported that cells overexpressing TLK1B or the KD recruited Rad9 in proximity of the DSB to much greater level than control cells, consistent with the specific interaction of TLK1B with Rad9. We postulated that the TLK1B-Rad9 interaction promotes and/or stabilizes their association with the DSB. Since it is the Rad17-RFC clamp-loader that locks the 9-1-1 complex onto damaged DNA [25], we wanted to test if the occupancy of Rad17 in proximity of the DSB was altered in KD-overexpressing cells. We reasoned that the recruitment of Rad17 to the DSB should not be affected by the KD, as there is no direct interaction of Rad17 with TLK1B, and further the binding of the clamp loader to DSBs precedes 9-1-1 assembly. On the other hand, it seemed possible that the phosphorylation of Rad9 cells could somehow affect the association or stability of the Rad17-RFC/9-1-1 complex with the DSB.

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