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-transcr ibed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I r epeat sequences located in the origin region cause defects in the repl ication and/or maintenance of amplified rDNA in vivo Fe(II)EDTA cleava ge footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by protei ns in extracts of Tetrahymena cells. Two classes of proteins that boun d the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of thes e, ds-TIBF, bound preferentially to duplex DNA and exhibited only mode rate specificity for Type I repeat sequences. In contrast, a single-st randed DNA-binding protein, ssA-TIBF, specifically recognized the A-ri ch strand of the Type I repeat sequence. Deletion of the 5' or 3' bord ers of the conserved sequence significantly reduced binding of ssA-TIB F. The binding properties of ssA-TIBF, coupled with genetic evidence t hat Type sequences function as cis-acting rDNA replication control ele ments in vivo, suggest a possible role for ssA-TIBF in rDNA replicatio n in Tetrahymena.