We previously published initial evidence that TLK1B protects a normal mammary cell line (MM3MG) from IR 8. This result is now reproduced in Fig. 1 in a more complete assay with serially diluted cells plated for clonogenic assays. We show that 90% of untransfected MM3MG were killed at 2 Gray (Gy), in contrast to only 55% of those transfected with TLK1B (P = 0.001 by one-tailed t-test). At 4 Gy, very few MM3MG cells survived, in contrast to 4% of cells expressing TLK1B (P = 0.0001). This confirmed that overexpression of TLK1B significantly increased their radioresistance.

We previously reported that TLK1B phosphorylates very well histone H3 at S¹⁰ but not the other core histones or H1 8. Interestingly, we also found that IR results in a loss of H3 phosphorylation, but the significance of this result was unclear. If TLK1 is the major kinase involved in a "chromosomal response" to DNA damage, then inhibition of TLK1 or TLK1B activity by IR through ATM is expected to result in a loss of phosphorylation of histone H3. To probe for the significance of the H3 dephosphorylation, we carried out a time course of recovery from IR and monitored the phosphorylation of H2AX and H3 in the MM3MG cells that overexpress TLK1B. Radiation-induced damage has been shown to result in rapid phosphorylation of histone H2AX at DSB sites 1516, while BRCA2, a protein that localizes to DSBs, was found to accumulate to sites of condensed chromatin colocalized with phosphorylated H3 17. As seen in Fig. 2, phosphorylation of H2AX is, as expected, very rapid after IR in both control and TLK1B cells. In the control, phosphorylation of H2AX remains elevated for at least 8 hr and slightly reduced after 16 hr of recovery. Further, phosphorylation of H3 drops drastically after IR and only recovers after 16 hr in the control. In TLK1B cells, basal phosphorylation of H3 is elevated at t = 0, as previously reported 8 and then decreases after IR (note that the Chk1 phosphorylation site is conserved in TLK1B). However, between 4 hr and 8 hr, the phosphorylation of H3 recovers completely. At the same time, phosphorylation of H2AX drops precipitously. This suggests that repair of DNA is much more rapid in TLK1B cells, if the phosphorylation state of histones H3 and H2AX and presumed remodeling of chromatin can be taken as an indicator of DSB repair.

If TLK1 and TLK1B are kinases that are largely responsible for the phosphorylation of H3 in unstressed conditions (excluding mitosis), and if activation of ATM following IR is responsible for inhibiting TLK1/B [as previously published; 34], then inhibition of ATM with wortmannin should prevent in large part the dephosphorylation of H3 following IR. Fig. 3 shows that this is indeed the case, since cells pretreated with wortmannin and subjected to IR showed only a very modest decrease in H3 phosphorylation throughout the time course of IR and recovery. Contrary to the expectation, phosphorylation of H2AX was not inhibited by wortmannin in either control or cells overexpressing TLK1B. This was somewhat surprising since ATM is believed to directly phosphorylate this histone after IR 18. However, very recently, it was demonstrated that ATR, which requires much higher concentrations of wortmannin for inhibition (0.1 mM), also phosphorylates H2AX after radiation, although with somewhat delayed kinetics 16.

To overexpress TLK1B, we used an episomal vector called BK-Shuttle 19. An important advantage is that these plasmids can be easily rescued from mammalian cells by the Hirt's supernatant protocol 19. Most stable cell lines carrying these vectors typically have hundreds of copies of the episomes, which have the typical structure of mini-chromosomes and thus behave like genomic DNA 2021. We previously showed that the extraction of episomes is a very simple and efficient protocol that yields very consistent amounts of plasmids that are linear with respect to cell numbers 19

In Fig. 4, we show an example of how these episomes can be used to study protection from IR-mediated DSBs. We have found that IR causes significant damage to the episomes, as shown by a large proportion of linearized molecules due to DSBs. Plasmids isolated from untreated cells migrate as a predominant supercoiled fast-migrating form and a relaxed circular form. Plasmids isolated from irradiated cells show a dose-dependent loss of the supercoiled form due to DSBs, which convert the plasmid to a linearized form (and some nicked circular). The conversion of closed (circular and supercoiled forms) to linearized plasmids can be exploited to monitor the activity of TLK1B. Since the episomes are assembled in typical chromatin, the function of TLK1B in repair (e.g., chromatin remodeling to produce ends suitable for ligation) would mirror its function on genomic DNA. Note that the intensities of the bands were quantified with an imaging program (ImageQuant), and that the sum of the bands (supercoiled, linear, or circular) was a constant, indicating very consistent yields of episomes.

The episomal vectors can be used as reporters for repair of DSBs and provide a useful system to test if this is the likely mechanism of radioresistance. Cells were irradiated and allowed to recover during an 8 hr time course (repair time), and plasmids were isolated for analysis by gel electrophoresis. In this experiment, however, the episomes were extracted from cells by alkaline lysis (essentially the same protocol used to extract plasmids from bacteria). Plasmids that are linearized with a DSB or plasmids with nicks are not recovered from alkaline lysis because of strand separation. In contrast, supercoiled/covalently closed plasmids are recovered from alkali treatment. Fig. 5 shows that IR causes an immediate and significant loss of supercoiled plasmids in control MM3MG cells containing the empty vector. Religation of the linear plasmids and formation of the supercoiled molecules does not occur until 4 to 8 hr of recovery from IR, and then, the recovery is only partial. In contrast, IR causes a similar loss of supercoiled molecules at t = 0 in MM3MG cells expressing TLK1B, but the recovery is very fast. At 2 hr (R2) the recovery of supercoiled molecules is almost complete and it is fully restored by 8 hr (R8). The recovery of the supercoiled forms is almost certainly due to the repair of pre-existing, damaged plasmids and not due to de novo synthesis. First, the repair time was too short to account for a large fraction of newly synthesized plasmids, and second, IR results in a dramatic arrest in DNA replication in normal cells 22, making it highly unlikely that any newly replicated plasmid was achieved. This experiment was repeated with identical results, indicating that the loss of supercoiled species after IR can be easily detected by alkaline lysis with great reproducibility.

To assess genomic repair, a modified terminal deoxytransferase (TdT) fill-in reaction was adopted. Briefly, the cells were irradiated (or not) to create genomic breaks, which were then processed with TdT and biotinylated dNTPs according to the manufacturer's protocol (see Methods). Macroscopic deposit of diaminobenzidine (DAB) at breakage sites was used to determine the extent of genomic damage and repair during a time course of recovery from IR (Fig. 6 and Table 1). No DAB deposits were visible in non-irradiated cells. In irradiated cells, 9–13 spots per cell were visible immediately after IR in both control and cells expressing TLK1B. Already at two hr of recovery from IR, in TLK1B cells the number of spots decreased by more than 50%, in contrast to control cells in which there was no appreciable recovery.

The direct way to probe the function of TLK1B in repair of a DSB is in vitro. Repair assays were carried out as described in Methods, based on a system that demonstrated a synergism between CAF-1 and Asf1 in a repair-coupled nucleosome assembly 23. If TLK1B increases the activity of Asf1 in vitro, then reactions supplemented with TLK1B should show a greater proportion of supercoiled topomers in addition to more ligated (closed) plasmid. Briefly, repair/nucleosome assembly was carried out on Bluescript plasmid that is linearized with EcoRI. We wished to monitor simultaneously: 1) Processing/ligation of the ends; and 2) superhelicity of the plasmid by the formation of nucleosomes on the template. Reactions contained nuclear extract, an energy mix, and additional purified TLK1B. The plasmid was then re-extracted and analyzed by electrophoresis in agarose gels. In Fig. 7A, linearized plasmids were incubated with cell extract +/- TLK1B, and repair was monitored during a time course. Note that the addition of TLK1B greatly stimulated the speed of the reaction in the formation of dimer molecules and the ligated/supercoiled forms of the plasmids, which already appear at 20 min instead of 40–60 min for extract without TLK1B. For shorter time points of incubation we used a more sensitive and quantitative method to monitor repair of the cut plasmids, i.e., a bacterial transformation assay. Linear plasmids do not transform bacteria, whereas repaired, covalently closed plasmids do. After 10 min of incubation with cell extract, in a typical experiment, we obtained 31 colonies vs. 260 for extract supplemented with exogenous TLK1B. Therefore, it appears that TLK1B improves the accessibility of the repair and ligation machinery to the free ends of the DNA or that it stimulates the deposition of histones [by Asf1; 11] to assemble chromatin. This lends strong support to our hypothesis that the principal mechanism of increased radioresistance is through more efficient repair of DSBs likely linked to chromatin remodeling.

In Fig. 7B, we show an assay of supercoiling activity, which is a measure of chromatin assembly. In this assay, the plasmid Bluescript (nearly 100% supercoiled, input) is used as a template for the deposition of core histones in the presence of nuclear extract and an energy mix. In the absence of histones, the extract causes the bacterially supercoiled form to convert to mostly relaxed forms due to endogenous topoisomerases (data not shown). However, after incubation in the presence of histones, the plasmid migrates as a series of discrete supercoiled forms due to the formation of nucleosomes, which decrease the linking number by one integer per nucleosome. The addition of recombinant TLK1B stimulated the formation of the more highly supercoiled forms, particularly the form that runs like bacterially supercoiled plasmid.

Normal breast epithelial cells, MM3MG, transfected or not with TLK1B were cultured as described in Li et al. 8.

Control MM3MG cells and the cells overexpressing TLK1B [MM3MG-TLK1B; 8] were harvested with PBS/EDTA and adjusted to 10,000 cells/tube in DMEM/10% FCS. Cells were irradiated in the Radiation department at LSUHSC with Elekta Precise linear accelerator at 6 MV. For each radiation dose levels (0 to 8 Gy), aliquots of serially diluted cells (100–5000) were plated on 6-well plates in triplicate. After a period of 10 days of incubation, the wells were rinsed with PBS and stained with crystal violet, and the colonies counted. The experiment was repeated thrice, and the results were expressed as the fraction of surviving cells compared to the number of colonies formed in the non-irradiated samples (plating efficiency).

The anti-histone H3 phosphorylated at Ser-10 and anti-histone H2AX phosphorylated at Ser-139 were from Upstate Cell Signaling (Lake Placid, NY). For Western blots, 30 μg of protein of each sample was separated on a 15% SDS/PAGE gel. The proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and incubated overnight with primary antiserum and for 1 hr with secondary antisera (1:1000 dil.). Finally, the membranes were washed three times and developed with Opti-4CN reagent (Bio-Rad, Hercules, CA).

Episomes were isolated from 2 × 10⁷ cells (90% confluent flasks) stably transfected with empty vector (BK-Shuttle) or vector carrying TLK1B. Two methods were used to extract the episomes: either the standard Hirt's supernatant protocol 35, or alkaline lysis. Briefly, the cells were resuspended in 0.1 ml TE, lysed at room temperature with solution 1 (0.2 ml of 0.2 M NaOH, 1% SDS), which was then neutralized with solution 2 (0.15 ml of 3 M K-Acetate/glacial acetic acid). After a brief centrifugation at 10,000 × g to remove insoluble material (including genomic DNA), remaining nucleic acids were extracted with Phenol/Chloroform (1:1), and precipitated with cold EtOH. The episomes were analyzed by gel electrophoresis on 1% agarose/TAE, and stained with EtBr.

To assess the repair of DNA damage in vivo, the modified TUNEL assay (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) was applied. MM3MG or TLK1B cells were grown to 50% confluence on tissue culture slides prior to exposing them to ionizing irradiation (20 Gy). After radiation, the cells were allowed to recover for varying times (0, 2, 8 hr). Subsequently, cells were fixed in 4% formalin/PBS and permeabilized in 0.2% Triton-X100/PBS. For labeling DNA breaks in situ, the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) was used according to the manufacturer's protocol. The biotinylated nucleotides incorporated at 3'OH ends were reacted with horseradish peroxidase labeled-streptavidin, which was then detected by diaminobenzidine (DAB). The nuclear DNA-labeled sites (brown spots) within each cell were counted under a light microscope (40 × magnification), and the average (± SD) number of DNA breaks per cell was calculated. At least 10 cells per dose were counted.

Nuclear extract from MM3MG cells was prepared as described in 36. Repair assays were carried out as described by Mello 23. Briefly, repair/nucloesome assembly was carried out on 0.1 μg of Bluescript plasmid (per reaction) that was linearized with EcoRI. We monitored simultaneously ligation of the ends and superhelicity of the plasmid by the formation of nucleosomes on the template. Reactions contain 20 μg of nuclear extract, 5 mM MgCl₂, 40 mM Hepes, pH7.8, 0.5 mM DTT, 4 mM ATP, 20 μM dNTPs, 4 mM phosphocreatine, 2 u of creatine phosphokinase, and additional recombinant TLK1B. After incubation at 37°C for the indicated amount of time, the reactions were deproteinized with phenol and the plasmid was re-precipitated with cold EtOH.

Nucloesomes assembly was carried out on 2 μg of Bluescript plasmid. Reactions contained 15 μg of MM3MG cell extract (which already contains sufficient amounts of topoisomerases), 5 mM MgCl2, 40 mM Hepes, pH 7.8, 0.5 mM DTT, 4 mM ATP, 20 μM dNTPs, 4 mM phosphocreatine, 2 u of creatine phosphokinase, and additional purified proteins (200 ng TLK1B and 2 μg supplemental HeLa histones). The reactions were incubated at 37°C for 0.5 hr. The plasmid was re- extracted with GeneClean III kit (Bio 101, Vista, CA), separated on an agarose gel and subsequently stained with EtBr.