5' C-rich telomeric overhangs are an outcome of rapid telomere truncation events

Abstract

A subset of human tumors ensures indefinite telomere length maintenance by activating a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). Most tumor cells of ALT origin share a constellation of unique characteristics, which include large stores of extra-chromosomal telomeric material, chronic telomere dysfunction and a peculiar enrichment in chromosome ends with 5' C-rich overhangs. Here we demonstrate that acute telomere de-protection and the subsequent DNA damage signal are not sufficient to facilitate formation of 5' C-overhangs at the chromosome end. Rather chromosome ends bearing 5' C-overhangs are a by-product of rapid cleavage events, processing of which occurs independently of the DNA damage response and is partly mediated through the XRCC3 endonuclease.

Figure 1. Telomere rapid deletions but not telomere dysfunction induce C-overhang formation in IMR90 cells

A. Western analysis of IMR90 cells expressing an empty vector or TRF2 deletion mutants. γ-tubulin serves as a loading control. A longer exposure to demonstrate the level of expression of endogenous TRF2 is shown in the lower panel. B. T-circle assays for 0.25, 0.5 and 1.0 μg of digested genomic DNA from IMR90 cells expressing the TRF2^ΔB mutant or vector control. KMST6 DNA was included as a positive control. The presence or absence of φ29 DNA polymerase is indicated. The gel was probed using radiolabeled (TTAGGG)₄ under denaturing conditions. C. 1D gels for digested genomic DNA (4 μg) from IMR90 cells stably expressing the indicated TRF2 mutants or vector control on days 3, 5, 8 and 11 of selection. The gels were probed for G- and C-rich telomeric DNA with radiolabeled (AATCCC)₅ or (TTAGGG)₄ probes respectively, under native (top) and denaturing (bottom) conditions. D. 2D gel analysis of digested genomic DNA (20 μg) from cells expressing the indicated TRF2 mutants on day 3 of selection. A radiolabeled (TTAGGG)₄ probe was used under native (top) and denaturing (bottom) conditions. Schematic diagrams of DNA species are shown on the left.

Figure 2. T-loop deletion events at overlengthened telomeres give rise to telomeric C-overhangs

A. 1D gels for restriction digested genomic DNA (4 μg) from cells overexpressing hTR or vector control at population doublings (PD) 10 or 250. The gels were probed for C-rich (top panels) versus G-rich (bottom panels) telomeric DNA under native (left panels) or denaturing (right panels) conditions as described in Figure 1. The amount of single stranded telomeric DNA in native gels was normalized against the total amount of telomeric DNA in denatured gels and expressed as a ratio indicated at the bottom. B. 2D gel analysis of genomic DNA (15 μg) from control or hTR overexpressing cells at PD 250. Gels were probed for both C- and G-strand specific telomeric DNA under native (top) and denaturing (bottom) conditions. The arrows indicate circular double-stranded telomeric DNA (see diagrams in Figure 1D).

Figure 3. T-circles and C-overhangs are enriched in Werner syndrome fibroblasts

A. 2D gel analysis of restriction digested genomic DNA (25 μg) from E6 and E7 expressing IMR90 or WRN syndrome fibroblasts. Gels were probed for both C- and G-strand specific telomeric DNA under native (top) and denaturing (bottom) conditions. Ethidium bromide staining of digested genomic DNA electrophoresed in the first dimension is indicated to demonstrate equivalent loading. B. T-circle assays for increasing concentrations (0.25, 0.5 and 1.0 μg) of digested genomic DNA from IMR90 or WRN syndrome fibroblasts. Digested DNA from KMST6 cells was included as a positive control. The presence or absence of φ29 DNA polymerase is indicated. The gel was probed as described in Figure 1B.

Figure 4. C-overhang formation is partially XRCC3-dependent

A. Immunoblots of whole cell lysates from E6 and E7 expressing IMR90 cells transduced with TRF2^ΔB or vector (pLPC) control retroviruses (day 4 of selection), and each further treated with either control or XRCC3 siRNAs for 72 hr. The blots were probed for TRF2 (top) or XRCC3 (bottom) expression. γ-tubulin serves as a loading control. B. Representative example of 1D gels for digested genomic DNA derived from the cells described in (A). Gels were probed under native (top) and denaturing (bottom) conditions with C- (left panels) or G-strand (right panels) specific telomeric oligonucleotide probes as described for Figures 1–3. C. Quantitation of three independent experiments as described in B. D. Proposed model for the generation of C-overhangs at the chromosome end. T-loop release partially mediated by XRCC3 and resulting in free circular ECTR and a shortened telomere is one requirement for C-overhang formation. Chromosome ends bearing a C-rich overhang do not necessarily bear a DNA damage signal.