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. 1994 Oct 25;22(21):4432–4440. doi: 10.1093/nar/22.21.4432

Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.

A R Umthun 1, Z Hou 1, Z A Sibenaller 1, W L Shaiu 1, D L Dobbs 1
PMCID: PMC308476  PMID: 7971273

Abstract

An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.

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Selected References

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