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

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.

Figure 1

Repair of linearized plasmids in cell extract. A) Preparation of Asf1-depleted cell extract. Depletion of Asf1A and Asf1B with respective siRNAs was monitored by western blot. B) Ligation of cohesive (EcoRI ends) and role of Asf1. The reaction was assembled as described in [15,3]. Plasmid cut with EcoRI was incubated with Asf1-depleted extract or control, to monitor end-joining repair coupled with nucleosome assembly. The positions of the linearized and closed/relaxed forms of the plasmid are marked. C) Ligation of EcoRI/EcoRV-cut plasmid. The reactions contained 5 μCi [α-³²P]dATP (0.1 mM final concentration) to label the plasmid by endogenous polymerases. In the left and right panels (autorad and EtBr), the stimulation of plasmid religation/supercoiling by added TLK1B is shown. The position of the linearized and ligated/supercoiled form is shown. Quantitation of the autorad in pixels (supercoiled form) is shown below each lane. D) Labeling by fill-in in Ku-depleted extract. The effect of TLK1B on end-labeling is shown in the absence of ligation. Quantitation of the autorad in pixels is shown below each lane.

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.

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.

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.

Figure 5

Association of Rad17 clamp-loader and Rad9 at chromatin adjacent a DSB. Conditions for ChIP were described in [11]. The occupancy of Rad17 (A) and Rad9 (B) adjacent to the DSB was monitored during a time course of adeno-HO infection, using primers puro1/puro2. The signals of the bands (quantitated with Image J) were normalized to that of the uncleaved DNA at the FABP1 locus. The average of the data and error-bars from 3 complete experiments are shown. C) HO-mediated death in control and TLK1B-KD-expressing cells. Cells were infected with adeno-HO, and either immediately collected or collected two days later. The cells were stained with Trypan blue and counted to monitor viability.

Figure 6

The phosphorylation of Rad9-S328 fluctuates during repair of DSBs. ES-Rad9-/- cells complemented with WT and S328A-Rad9 were irradiated (5 Gy), and immediately processed (t = 0) or allowed to recovery for the indicated hours. The blots were sequentially probed with an antiserum for P-Rad9 and for actin.

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].