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. 2006 Oct 20:7:37.
doi: 10.1186/1471-2199-7-37.

TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1

Siddhartha P Sen  1 , Arrigo De Benedetti
Affiliations

TLK1B promotes repair of UV-damaged DNA through chromatin remodeling by Asf1

Siddhartha P Sen et al. BMC Mol Biol. .

Abstract

Background: The mammalian protein kinase TLK1 is a homologue of Tousled, a gene involved in flower development in Arabidopsis thaliana. The function of TLK1 is not well known, although knockout of the gene in Drosophila, or expression of a dominant negative mutant in mouse mammary cells causes loss of nuclear divisions and chromosome mis-segregation. TLK1B is a splice variant of TLK1 and it confers radioresistance in a normal mammary mouse cell line possibly due to increased chromatin remodeling capacity, but the mechanism of resistance remains to be fully elucidated.

Results: We now show that TLK1B also affords protection against UV radiation. We find that nuclear extracts isolated from TLK1B-containing mouse cells promote more efficient chromatin assembly than comparable extracts lacking TLK1B. TLK1B-containing extracts are also more efficient in repair of UV-damaged plasmid DNA assembled into nucleosomes. One of the two known substrates of TLK1 (or TLK1B) is the histone chaperone Asf1, and immuno-inactivation experiments suggest that TLK1B increases UV-repair through the action of Asf1 on chromatin assembly/disassembly.

Conclusion: Our studies provide evidence for TLK1B-mediated phosphorylation of Asf1 triggering DNA repair. We suggest that this occurs via Asf1-mediated chromatin assembly at the sites of UV damage.

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Figures

Figure 1
Figure 1
Survival curves after UV irradiation. Percentage survival of MM3MG, MM3-TLK1B and MM3-KD cells after exposure to 1, 1.5, 2, 3, 4 and 6 J/m2 of UV are shown. The average of triplicate experiments is shown. The D0 values were 3.5 J/m2 for the MM3-TLK1B cells, 2 J/m2 for the MM3MG cells and 1.2 J/m2 for the MM3-KD cells respectively.
Figure 2
Figure 2
Repair of genomic DNA in MM3MG and MM3-TLK1B cell lines following UV-radiation. (A) A slot blot assay carried out to detect pyrimidine dimmers (CPDs) using a monoclonal antibody. Unirradiated DNA (slot 1) was used as a control. (Representative of three experiments). (B) Graphical representation of the data shown in figure 2A. Average of three experiments is shown. The X-axis denotes the hours of recovery following UV-irradiation, while the Y-axis represents the CPDs remaining in terms of percentage (0 hrs: 100%; No UV control: 0%).
Figure 3
Figure 3
Repair of episomes in intact irradiated cells. MM3MG cells (stably transfected with the empty BK-shuttle vector) and MM3-TLK1B cells were exposed to 5 J/m2 of UV (0 hrs) and allowed to recover for 0, 2, 4, 8 and 12 hours. Episomal vector extracted from both cell lines (control as well as UV-irradiated) at shown time-points was either mock-treated (-) or digested with T4 endonuclease V (+).
Figure 4
Figure 4
Phosphorylation of H3-Ser 10 after UV-irradiation. MM3MG and MM3-TLK1B cells were grown to 80% confluence prior to UV-radiation (5 J/m2). Cells were harvested at different times (0, 2, 6 and 8 hrs) after irradiation and lysed in RIPA buffer. Equal amount of protein of each sample was loaed on a 15% SDS-PAGE gel and electrophoresed. Blots were probed with phospho-Histone H3 (Ser 10) antibody. Equal loading of proteins was confirmed by blotting for tubulin. These blots are representative of two separate experiments.
Figure 5
Figure 5
(A) MNase digestion of pBluescript assembled into nucleosomes. Lane 1:Marker; Lanes 2–6: MNase treatment for 0, 2, 4 and 8 minutes respectively. (B) MNase digestion of naked plasmid.
Figure 6
Figure 6
(A) In vitro DNA repair assay on nucleosomal template showing incorporation of [α]32P-dATP over a time course by MM3MG and MM3-TLK1B extracts. The left panel shows unirradiated DNA while the right panel shows UV-treated plasmid DNA. The numbers at the top of the gel indicate the time for which the plasmid was incubated in the extract, ranging from 5 to 30 minutes. The figure shown is a representative of five independent experiments. (B) Graphical representation of the data shown in figure 6(A). The maximum repair in the MM3MG (control) cell line has been set at 100% (30 minute time point) and the standard-deviations from three independent experiments have been used for the error bars.
Figure 7
Figure 7
A Chromatin assembly (supercoiling assay) of bluescript plasmid with MM3MG extract supplemented with Asf1 and/or TLK1B. Input: Fully supercoiled plasmid prior to incubation in nuclear extract. Plasmid was assembled into chromatin by incubation (for 1 hour at 37°C) with extract, energy mix and the specified components indicated. The figure is a representative of three separate experiments. The densitometric profile for each condition is shown. Note the increase in intensity of band 7 in lane 6. B Chromatin assembly (supercoiling assay) of bluescript plasmid in the presence of TLK1-antiserum (MM3MG extracts). Lane 1: Input plasmid; Lane 2: Plasmid + extract; Lane 3: Plasmid + extract + TLK1-antiserum; Lane 4: Plasmid + extract + pre-immune serum. Lane 5: Plasmid + extract + TLK1-antiserum + recombinant TLK1B re-addition. The assay shown is a representative of two independent experiments. (MM3MG and MM3-TLK1B extracts gave similar results).
Figure 8
Figure 8
Chromatin assembly (supercoiling assay) of undamaged (lanes 2 and 4) and UV-damaged (lanes 3 and 5) pBluescript by MM3MG (lanes 2 and 3) and MM3-TLK1B (lanes 4 and 5) extracts. Note the presence of more supercoiled topomers (Forms 1,2 and 3) in lane 5 as compared to lane 3.
Figure 9
Figure 9
(A) In vitro repair assay using increasing amounts of recombinant Asf1b. Lanes 1–3: Repair by MM3MG extracts; Lanes 4–6: Repair by MM3-TLK1B extracts. Asf1b increases from 0 to 200 ng as shown on the top. (B) Graphical representation of the data in figure 9A. The maximum repair in the MM3MG (control) cell line has been set at 100%(Lane 3, 200 ng Asf1b) and the standard-deviations from three independent experiments have been used for the error bars. (C) Western blot of MM3MG and MM3-TLK1B extracts using α-Asf1b antibody.
Figure 10
Figure 10
(A)In vitro repair assay in the absence (lane 1) or presence (lanes 2–5) of Asf1-antiserum (MM3MG extracts). The numbers below each lane indicate the amount of radiolabel incorporated as percentage of control (lane 1), while the numbers at the top indicate the period of plasmid incubation in the presence of MM3MG nuclear extract. MM3MG and MM3-TLK1B extracts gave similar results. (Representative of two separate experiments). (B) In vitro repair assay in the presence of Asf1-antiserum (lane 2) followed by complementation with recombinant Asf1b protein (lanes 3 and 4).
Figure 11
Figure 11
In vitro repair assay in the presence of TLK1-antiserum (lanes 3, 4 and 5), followed by complementation with recombinant TLK1B protein (lanes 4 and 5). Numbers at the bottom indicate the amount of radiolabel incorporated as percentage of control (lane 1). MM3MG and MM3-TLK1B extracts gave similar results. (Representative of two separate experiments).
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References

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