The dhfr ori beta-binding protein RIP60 contains 15 zinc fingers: DNA binding and looping by the central three fingers and an associated proline-richregion

Initiation of DNA replication occurs with high frequency within ori beta, a short region 3' to the Chinese hamster dhfr gene, Homodimers of RIP60 (rep lication initiation-region protein 60 kDA) purified from nuclear extract bi nd two ATT-rich sites in ori beta and foster the formation of a twisted 720 bp DNA loop in vitro, Using a one hybrid screen in yeast, we have cloned t he cDNA for human RIP60, RIP60 contains 15 C2H2 zinc finger (ZF) DNA bindin g motifs organized in three clusters, termed hand Z1 (ZFs 1-5), hand Z2 (ZF s 6-8) and hand Z3 (ZFs 9-15), A proline-rich region is located between han ds Z2 and Z3, Gel mobility shift and DNase I footprinting experiments show hands Z1 and Z2 independently bind the ori beta RIP60 sites specifically, b ut with different affinities, Hand Z3 binds DNA, but displays no specificit y for RIP60 sites. Ligation enhancement, DNase I footprinting, and atomic f orce microscopy assays show that hand Z2 and a portion of the associated pr oline-rich region is sufficient for protein multimerization on DNA and DNA looping in vitro, Polyomavirus origin-dependent plasmid replication assays show RIP60 has weak replication enhancer activity, suggesting that RIP60 do es not harbor a transcriptional transactivation domain. Because vertebrate origins of replication have no known consensus sequence, we suggest that se quence-specific DNA binding proteins such as RIP60 may act as accessory fac tors in origin identification prior to the assembly of pre-initiation compl exes.