REGULATION OF RDNA REPLICATION IN TETRAHYMENA

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The precise control of DNA replication is a prerequisite for faithful transmission of genetic information in all dividing cells. The molecular mechanisms that regulate DNA replication in eukaryotic cells are not understood. The goal of this project is to investigate the molecular details of DNA replication control in eukaryotes, by focusing on the replication of the amplified rRNA genes (rDNA) in the single-celled protozoan, Tetrahymena thermophila. The proposed experiments investigate the contrdl of rDNA replication in two ways. One is the delineation of cis-acting DNA sequence signals required for accurate and efficient replication of the rDNA. The other is the identification of proteins or other trans-acting factors that act at these sites. Specifically, cis- acting control regions will be identified by: 1) determining the nucleotide sequence changes responsible for rDNA replication defects in two mutant Tetrahymena strains; 2) mapping precisely the in vivo origins of replication in these and additional previously characterized replication mutants; and 3) assaying the replication properties of altered rDNA constructs both in vitro, using cell- free replication extracts, and in vivo, by microinjection of Tetrahymena cells. The trans-acting factors that mediate regulation of rDNA replication will be investigated by: 1) identifying specific DNA binding proteins that interact with the origin of replication and other critical control sites identified above; and 2) testing the possibility that specific RNA transcripts are involved in regulating rDNA replication and copy number. Analysis of the molecular mechanisms that ensure faithful DNA replication is essential from understanding the fundamental processes that regulate normal cell growth and division, and should provide use full insights into the causes of aberrant growth control.