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Oxidative guanine base damage regulates human telomerase activity

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Abstract

Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative alterations in telomere length and integrity. Here we examined how the common oxidative lesion 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG) regulates telomere elongation by human telomerase. When 8-oxoG is present in the dNTP pool as 8-oxodGTP, telomerase utilization of the oxidized nucleotide during telomere extension is mutagenic and terminates further elongation. Depletion of MTH1, the enzyme that removes oxidized dNTPs, increases telomere dysfunction and cell death in telomerase-positive cancer cells with shortened telomeres. In contrast, a preexisting 8-oxoG within the telomeric DNA sequence promotes telomerase activity by destabilizing the G-quadruplex DNA structure. We show that the mechanism by which 8-oxoG arises in telomeres, either by insertion of oxidized nucleotides or by direct reaction with free radicals, dictates whether telomerase is inhibited or stimulated and thereby mediates the biological outcome.

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Figure 1: 8-oxodGTP is a telomerase chain terminator.
Figure 2: Telomerase preferentially incorporates 8-oxodGTP opposite rA.
Figure 3: Cells with shortened telomeres are hypersensitive to oxidized dNTPs.
Figure 4: Oxidized dNTPs induce telomere defects in cells with shortened telomeres.
Figure 5: Telomerase elongation of primers with a terminal 8-oxoG.
Figure 6: 8-oxoG restores telomerase activity on quadruplex folded overhangs.
Figure 7: 8-oxoG increases the dynamics of telomeric GQ structures.

Change history

  • 14 December 2016

    In the version of this article originally published, the Online Methods section erroneously identified the probe used for immunofluorescence and in situ hybridization assays. The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

We thank S. Watkins and C. St. Croix (University of Pittsburgh Center for Biological Imaging) for assistance with fluorescence imaging, S. Uttam for help with statistical analyses, T. Moiseeva for assistance with the annexin V and propidium iodide FACS assay and R. O'Sullivan and B. Van Houten for critical reading of the manuscript. We also thank T. Cech and A. Zaug (University of Colorado) for reagents and assistance with the telomerase assays and R. O'Sullivan (University of Pittsburgh) for generously providing the HeLa VST and HeLa LT cell lines. This work was supported by NIH grants R01ES022944, R21AG045545 and R21ES025606 (to P.L.O.), American Cancer Society RSG-12-066-01-DMC and NIH 1DP2GM105453 (to T.L., G.K. and S.M.), NIH grant R00ES024431 (to B.D.F.), and NIH grant CA148629 and the Abraham A. Mitchell Distinguished Investigator fund (to R.W.S.). This project used the UPCI CTIF and CF, which are supported in part by award P30CA047904.

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Contributions

E.F., J. Lormand and A.B. performed biochemical and cellular experiments, analyzed the data and prepared figures. H.-T.L. and G.S.K. performed all the smFRET studies. J. Li and R.W.S. provided lentiviruses for MTH1 depletion experiments, and R.W.S. provided helpful discussions. E.F., B.D.F., S.M. and P.L.O. designed experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Patricia L Opresko.

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R.W.S. is a scientific consultant for Trevigen, Inc. The other authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 8-oxodGTP inhibits telomerase activity.

(a) Telomerase reactions were conducted using (TTAGGG)3 substrate (oligo #1 in Supplementary Table 1) and contained high dNTPs (lanes 1 - 4) or cellular dNTPs (lanes 5 - 8) and 0.3 μM 32P-α-dGTP. Natural dGTP and 8-oxodGTP were mixed at 1:0, 1:1, and 0:1 ratios to maintain a total concentration of 500 μM or 5.2 μM of unlabeled guanine nucleotide. Lanes marked with 0* contained 0.3 μM 32P-α-dGTP, and lacked unlabeled dGTP. Products were separated on polyacrylamide denaturing gels. The LC was a 32P end-labeled 18-mer oligonucleotide. Numbers with plus sign indicate number of telomeric repeats added. (b) Products were quantitated and normalized to the LC, and used to calculate processivity (top graph) and relative activity (bottom graph). Bars represent the mean and s.d. from three independent reactions. Asterisks, ** p < 0.01, *** p < 0.001 based on two-tailed Student’s t test.

Supplementary Figure 2 Elongation termination after 8-oxodGTP is not due to inhibition of telomerase translocation.

Telomerase reactions were conducted using (GGTTAG)3 (oligonucleotide 2 in Supplementary Table 1), and contained cellular dNTPs (37 μM dTTP, 24 μM dATP, 29 μM dCTP, 5.2 μM dGTP) and 0.3 μM 32P-α-dTTP. dGTP and 8-oxodGTP were mixed at 1:0, 1:1, and 0:1 ratios to maintain a total concentration of 5.2 μM of unlabeled guanine nucleotide. Products were separated on polyacrylamide denaturing gels. The LC was a 32P end-labeled 18-mer oligonucleotide. Numbers with plus sign indicate number of repeats added.

Supplementary Figure 3 MTH1 depletion generates DNA damage in HeLa cell lines with very short or long telomeres.

(a) Telomere lengths were confirmed by Southern blotting of telomeric restrictions fragments. The DNA ladder is shown. UD indicates undigested genomic DNA and D indicates digested genomic DNA. (b) Cell lysate was immuno-stained for catalase and SOD expression prior to lentiviral infection. (c) Cells were immuno-stained with an 8-oxoG antibody (green) three days after infection with lentivirus expressing a non-targeting shRNA (scr) or two different shRNAs targeting MTH1 (sh4 or sh5). Nuclei are indicated by DAPI staining (blue). Exposure times and image settings were equivalent for the sh4 and sh5, compared to the scr control. (d) Cells were stained for telomeric DNA by FISH (green) and immuno-stained with a TRF2 antibody (red) three days after lentiviral infection. Nuclei are indicated by DAPI. (e) The average number of TIFs per cell nuclei was calculated after three days post infection with lentivirus. Bars represent the mean ± sd from 3 independent experiments (100 – 150 cells per condition). * p<0.01 based on One-way ANOVA with Tukey’s honest significance difference. (f) Prolong cell culturing partly restores MTH1 expression. Cell lysate was immuno-stained for MTH1 following 138 days after lentiviral infection, which corresponds to population doubling (PD) numbers or HeLa VST of 151 (scr and sh5) or 139 (sh4) and for HeLa LT of 140 (scr and sh5) and 135 (sh4).

Supplementary Figure 4 Detection of cell death in MTH1-depleted cells.

(a and b) Apoptosis was measured by annexin V staining and flow cytometry in HeLa VST or HeLa LT 6 (a) and 9 (b) days after infection with lentivirus expressing a non-targeting shRNA (scr) or different shRNAs against MTH1 (sh4 and sh5). Numbers above red box indicate total annexin V positive cells. Bar graphs show the mean ± s.d. of 4 replicates. ****p<0.0001, by two-tailed Student’s t test. By 9 days post-infection no significant increase in apoptosis is detected in either cell line. (c) MTH1 depletion triggers minimal apoptosis in BJ-hTERT and BJ fibroblasts compared to controls, 3 days after lentivirus infection, compared to controls, as measured by annexin V by flow cytometry.

Supplementary Figure 5 Telomerase activity modulates sensitivity to MTH1 depletion

(a) Immunoblot with antibodies against MTH1 or actin. The percent MTH1 expression relative to the controls are shown. (b) Cells were immuno-stained with an 8oxoG antibody (green) 3 days after infection with lentivirus expressing a non-targeting shRNA (scr) or two different shRNAs targeting MTH1 (sh4 or sh5). Nuclei are indicated by DAPI staining (blue). Exposure times and image settings were equivalent for the sh4 and sh5, compared to the scr control. (c) BJhTERT and BJ cells were fixed 3 days after lentiviral infection and stained for SA-beta-gal activity. The percentage of positive cells is indicated on the bar graph. (d) Whole cell lysates (10μg) were immunoblotted with anti-p53 and anti-actin antibody as a loading control 3 days after lentiviral infection. (e) MTH1 knock down impairs BJ-hTERT proliferation as compared to BJ fibroblasts. DIC images were captured 12 days following lentiviral infection with a 10x objective.

Supplementary Figure 6 Telomerase activity on terminal primers with 8-oxoG opposite rA.

(a) Telomerase reactions were conducted using primers containing three TTAGGG repeats terminating in GGG, GGGoxoG, or GGGToxoG (Supplemental Table 1, oligos #3, 9 and 10). Reactions contained cellular dNTP concentrations and 0.3 μM 32P-α-dGTP. Products were separated on denaturing gels. The LC was a 32P end labeled 36-mer oligonucleotide. Numbers with plus sign indicate number of added repeats. (b) and (c), Products were normalized to the LC, and used to calculate processivity (b) and relative activity (c). Bars represent the mean ± s.d. from three independent reactions. (d) Telomerase reactions contained high dNTPs (lane 1) or 2.9 μM dGTP (lanes 2-4) and 0.3 μM 32P-α-dGTP. Products were separated on polyacrylamide denaturing gels. The LC was 32P end-labeled 18-mer oligonucleotide. Bands representing addition (+) or subtraction (-) of telomeric repeats are indicated. Product size and corresponding sequence are shown, red = radiolabeled G insertion. (e) Products were quantitated and normalized to the LC, and used to calculate relative activity. Bars represent the mean and s.d. from three independent reactions.

Supplementary Figure 7 Telomerase-associated nuclease removes a primer terminal 8-oxoG.

(a) Telomerase preparations lack contaminating exonuclease activity. 8-oxoG substitutions in the 4R substrate are indicated in red “Go” (oligos 6-8 in Supplementary Table 1). Reactions (20 μl) contained 19.5 pmol primer and 0.5 pmol 32P end-labeled primer for a final total concentration of 1 μM in 1x human telomere buffer. The dNTPs were omitted. Reactions were initiated by adding 6 μl telomerase extract and incubated for 60 min at 30oC, and then run on polyacrylamide denaturing gels. (b) Telomerase reactions were conducted using primers containing three or four TTAGGG repeats, with a 3’ terminal G or 8-oxoG (oligos 3, 4, 6 and 7 Supplementary Table 1). Reactions contained high dNTPs (lane 1) or 2.9 μM dGTP (lanes 2-5) and 0.3 μM 32P-α-dGTP. Products were separated on polyacrylamide denaturing gels. The LC was 32P end-labeled 18-mer oligonucleotide. Bands representing addition (+) or subtraction (-) of telomeric repeats are indicated for 3 repeat (left) and 4 repeat (right) substrate. Product size and corresponding sequence are shown, red = radiolabeled G insertion. (c) Products were quantitated and normalized to the LC, and used to calculate relative activity. Bars represent the mean and s.d. from three independent reactions. Asterisks, ** p < 0.01, *** p < 0.001 base on two-tailed Student’s t test.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1796 kb)

Supplementary Data Set 1

Uncropped gels and blot images from main figures (PDF 22741 kb)

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Fouquerel, E., Lormand, J., Bose, A. et al. Oxidative guanine base damage regulates human telomerase activity. Nat Struct Mol Biol 23, 1092–1100 (2016). https://doi.org/10.1038/nsmb.3319

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