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Molecular and Cellular Biology, February 2009, p. 703-713, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.00603-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Unusual Telomeric DNAs in Human Telomerase-Negative Immortalized Cells{triangledown} ,{dagger}

Akira Nabetani and Fuyuki Ishikawa*

Laboratory of Cell Cycle Regulation, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan

Received 14 April 2008/ Returned for modification 3 June 2008/ Accepted 10 November 2008

A significant fraction of human cancer cells and immortalized cells maintain telomeres in a telomerase-independent manner called alternative lengthening of telomeres (ALT). It has been suggested that ALT involves homologous recombination that is expected to generate unique intermediate DNAs. However, the precise molecular mechanism of ALT is not known. To gain insight into how telomeric DNAs (T-DNAs) are maintained in ALT, we examined the physical structures of T-DNAs in ALT cells. We found abundant single-stranded regions in both G and C strands of T-DNAs. Moreover, two-dimensional gel electrophoreses and native in-gel hybridization analyses revealed novel ALT-specific single-stranded T-DNAs, in addition to previously reported t-circles. These newly identified ALT-specific T-DNAs include (i) the t-complex, which consists of highly branched T-DNAs with large numbers of internal single-stranded portions; (ii) ss-G, which consists of mostly linear single-G-strand T-DNAs; and (iii) ss-C, which consists of most likely circular single-C-strand T-DNAs. Cellular-DNA fractionation by the Hirt protocol revealed that t-circles and ss-G exist in ALT cells as extrachromosomal and chromatin-associated DNAs. We propose that such ALT-specific T-DNAs are produced by telomere metabolism specific to ALT, namely, homologous recombination and the rolling-circle replication mechanism.

* Corresponding author. Mailing address: Laboratory of Cell Cycle Regulation, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, G-405, Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan. Phone: (81) 75-753-4195. Fax: (81) 75-753-4197. E-mail: fishikaw{at}lif.kyoto-u.ac.jp

{triangledown} Published ahead of print on 17 November 2008.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, February 2009, p. 703-713, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.00603-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oganesian, L., Karlseder, J. (2009). Telomeric armor: the layers of end protection. J. Cell Sci. 122: 4013-4025 [Abstract] [Full Text]  


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