Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA.
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.
Umthun
A R
AR
Department of Zoology and Genetics, Iowa State University, Ames 50011.
Hou
Z
Z
Sibenaller
Z A
ZA
Shaiu
W L
WL
Dobbs
D L
DL
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017124:Conserved Sequence; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D016054:DNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D004275:DNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin; D016808:Tetrahymena thermophila / Q000235:genetics*
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D017665:Hydroxyl Radical; D004262:DNA Restriction Enzymes