A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA

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Date
1995
Authors
Hou, Zhen
Umthun, Angela
Dobbs, Drena
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Zoology and Genetics
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Zoology and GeneticsMolecular, Cellular and Developmental BiologyGenetics
Abstract

Type I repeat sequences are evolutionarily conserved sequence elements found in the replication origin and transcriptional promoter region of the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single-stranded DNA binding protein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence. Quantitative binding competition experiments performed with purified ssATIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3' flanking region: addition of the 3' flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent K& = 3.0 x 10~10 M). A mutation in the ssA-TIBF binding site previously shown to be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro. ssA-TIBF also binds with high affinity to a copy of the Type I repeat sequence within the essential promoter region defined by in vitro transcription assays. The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region. The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.

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Reprinted (adapted) with permission from A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA, Zhen Hou, Angela R. Umthun, and Drena L. Dobbs, Biochemistry 1995 34 (14), 4583-4592, DOI: 10.1021/bi00014a011. Copyright 1995 American Chemical Society.

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