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Molecular and Cellular Biology, October 1999, p. 6699-6709, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin

Yukiko Okuno,1 Hiroyasu Satoh,2 Mariko Sekiguchi, and Hisao Masukata3,*

Department of Biology, Graduate School of Science, Osaka University, Toyonaka,1 and Precursory Research for Embryonic Science and Technology System, Japan Science and Technology Corporation,3 Osaka 560-0043, and Department of Molecular Biology, School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602,2 Japan

Received 23 March 1999/Returned for modification 13 May 1999/Accepted 23 June 1999

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.

* Corresponding author. Mailing address: Department of Biology, Graduate School of Science, Osaka University, 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Phone: 81-6-6850-5432. Fax: 81-6-6850-5440. E-mail: masukata{at}bio.sci.osaka-u.ac.jp.

Molecular and Cellular Biology, October 1999, p. 6699-6709, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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