Initiation of DNA synthesis is a critical control point in the mammalian cell cycle. Strand-specific hybridization studies with Okazaki fragments have mapped an origin of bidirectional DNA replication (OBR) to a 950 bp region 3' to the Chinese hamster dihydrofolate reductase (dhfr) gene. The dhfr origin region contains a fragment of stably bent DNA that binds multiple cellular factors, including the novel DNA binding protein, RIP60, that copurifies with the ATP-dependent DNA helicase, RIP100. This proposal is directed toward understanding the role that RIP60/RIP100 and other cellular factors play in regulating initiation of DNA synthesis at the dhfr origin. RIP60 and RIP100 will be purified in quantity by an established protocol; purified protein will be used for protein sequencing, biochemical analysis of RIP60/RIP100 function, and the production of specific antibodies. Protein sequence data will be used to identify cDNA clones for RIP60 and RIP100. cDNA clones and antibodies to RIP60 and RIP100 will be used to study the expression and cellular location of these factors throughout the cell cycle. The nature of the association between RIP60 and RIP100, in the presence and absence of specific and nonspecific DNA sequences, will be studied by scanning transmission electron microscopy, gel filtration, and sedimentation techniques. The helicase activity of RIP100 will be studied in regard to ATPase activity, ability to unwind dsDNA with and without RIP60 binding sites, and ability to unwind ds plasmids containing origin sequences. The effect of single-stranded DNA binding proteins (e.g. SSP, gene 32, and RF-A) on these reactions will be evaluated. The occupation of RIP60 and other protein binding sites in vivo will be examined as a function of the cell cycle. A stable transfection assay will be used to study origin function after integration into new positions in the genome. An emphasis will be placed on studying the role of bent DNA in origin function. The identification of DNA sequences required for origin function, and the purification of proteins involved in origin activation, will provide fundamental information concerning the regulation of cellular proliferation during growth, differentiation, and disease.
