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Originally published In Press as doi:10.1074/jbc.M106162200 on September 27, 2001

J. Biol. Chem., Vol. 276, Issue 48, 45417-45426, November 30, 2001
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Cloning and Biochemical Analysis of the Tetrahymena Origin Binding Protein TIF1
COMPETITIVE DNA BINDING IN VITRO AND IN VIVO TO CRITICAL rDNA REPLICATION DETERMINANTS*

Swati SahaDagger , Audrey Nicholson, and Geoffrey M. Kapler§

From the Department of Medical Biochemistry and Genetics, Texas A & M Health Science Center, College Station, Texas 77843-1114

Cis-acting type I elements regulate the initiation of DNA replication, replication fork movement, and transcription of the Tetrahymena thermophila rDNA minichromosome and are required for cell cycle-controlled replication and developmentally programmed gene amplification. Previous studies identified three in vitro single-stranded type I element binding activities that were proposed to play distinct roles in replication control. Here we describe the cloning of one of these genes, TIF1, and we provide evidence for its association with type I elements in vivo. Furthermore, we show that TIF1 interacts (in vitro and in vivo) with pause site elements (PSE), which co-localize with replication initiation and fork arrest sites, and are shown to be essential. The in vivo accessibility of PSE and type I elements to potassium permanganate suggests that origin regions are frequently unwound in native chromatin. TIF1 contains sequence similarity to the Solanum tuberosum single strand-specific transcription factor, p24, and a related Arabidopsis protein. Antisense inhibition studies suggest that TIF1 competes with other proteins for PSE and type I element binding. TIF1 displays a marked strand bias in vivo, discriminating between origin- and promoter-proximal type I elements. We propose that this bias selectively modulates the binding of a different subset of proteins to the respective regulatory elements.

* This work was supported by National Institutes of Health Grant GM53572.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF417528.

Dagger Current address: Dept. of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA 22908.

§ To whom correspondence should be addressed. Tel.: 979-847-8690; Fax: 979-847-9481; E-mail: gkapler@tamu.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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