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Molecular and Cellular Biology, November 2004, p. 9948-9957, Vol. 24, No. 22
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.22.9948-9957.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

Anthony J. Cesare and Jack D. Griffith*

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Received 20 July 2004/ Returned for modification 16 August 2004/ Accepted 26 August 2004

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.

* Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, Rm. 11-119, CB 7295, Mason Farm Rd., University of North Carolina, Chapel Hill, NC 27599-7295. Phone: (919) 966-2151. Fax: (919) 966-3015. E-mail: jdg{at}med.unc.edu.

Molecular and Cellular Biology, November 2004, p. 9948-9957, Vol. 24, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.22.9948-9957.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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