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Molecular and Cellular Biology, September 2001, p. 5753-5766, Vol. 21, No. 17
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.17.5753-5766.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

In Vitro Reconstitution of the End Replication Problem

Rieko Ohki,1 Toshiki Tsurimoto,2 and Fuyuki Ishikawa1,*

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501,1 and Nara Institute of Science and Technology, Ikoma, Nara 630-0101,2 Japan

Received 16 March 2001/Returned for modification 25 April 2001/Accepted 24 May 2001

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal ~500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.

* Corresponding author. Mailing address: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Phone: 81 45 924 5711. Fax: 81 45 924 5831. E-mail: fishikaw{at}bio.titech.ac.jp.

Molecular and Cellular Biology, September 2001, p. 5753-5766, Vol. 21, No. 17
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.17.5753-5766.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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