Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

Abstract

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.

FIG. 1.

Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ-³²P](CCCTAA)₆ probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ-³²P](CCCTAA)₆, and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.

FIG. 2.

Extrachromosomal DNA circles from GM847 and GM847-Tert cells. EMs of circular DNA molecules observed in the telomere-enriched fractions from GM847 and GM847-Tert cells. DNA was prepared for EM by surface spreading with cytochrome c and rotary shadowcasting. Images are shown in negative contrast. Circle lengths are 22.1, 8.9, 6.2, 3.9, 3.0, 1.6, and 0.8 kb for A through G, respectively. Bar is equivalent to 1 kb.

FIG. 3.

Summary of extrachromosomal circle (t-circles) and t-loop observations by EM. (A and B) Distribution of extrachromosomal DNA circle size as a percentage of scored molecules observed in the telomere-enriched fractions from GM847 (A) and GM847-Tert (B) cells. (C and D) Distribution of the loop portion (C) and total t-loop length (D) as a percentage of scored molecules from the psoralen cross-linked, telomere-enriched fractions from GM847 and GM847-Tert cells.

FIG. 4.

2D PFGE of TRFs from ALT and non-ALT cells. Total DNA (20 μg) was digested with HinfI/HaeIII and then separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ-³²P](CCCTAA)₆ probe and was visualized using a PhosphorImager. The black and white arrows indicate linear- and circular-form DNA, respectively.

FIG. 5.

2D PFGE of Hirt supernatant from ALT and non-ALT cells. An equal number of cells (10⁷) were Hirt precipitated, and the supernatant was separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ-³²P](CCCTAA)₆ probe and was visualized by PhosphorImager. The black and white arrows indicate supercoiled- and open-circular-form DNA, respectively.

FIG. 6.

t-loops from GM847 and GM847-Tert cells. EMs of t-loop molecules observed in the telomere-enriched fractions of GM847 and GM847-Tert DNA. DNA was prepared for EM as described for Fig. 2 and is shown in negative contrast. Loop and tail sizes are 19.0 and 8.4, 4.8 and 9.6, and 7.2 and 9.5 kb for panels A through C, respectively. Bar, 2 kb.