The dhfr oriβ-binding protein RIP60 contains 15 zinc fingers: DNA binding and looping by the central three fingers and an associated proline-rich region

Initiation of DNA replication occurs with high frequency within oriβ, a short region 3′ to the Chinese hamster dhfr gene. Homodimers of RIP60 (replication initiation-region protein 60 kDA) purified from nuclear extract bind two ATT-rich sites in oriβ and foster the formation of a twisted 720 bp DNA loop in vitro. Using a one hybrid screen in yeast, we have cloned the cDNA for human RIP60. RIP60 contains 15 C(2)H(2) zinc finger (ZF) DNA binding motifs organized in three clusters, termed hand Z1 (ZFs 1–5), hand Z2 (ZFs 6–8) and hand Z3 (ZFs 9–15). A proline-rich region is located between hands Z2 and Z3. Gel mobility shift and DNase I footprinting experiments show hands Z1 and Z2 independently bind the oriβ RIP60 sites specifically, but with different affinities. Hand Z3 binds DNA, but displays no specificity for RIP60 sites. Ligation enhancement, DNase I footprinting, and atomic force microscopy assays show that hand Z2 and a portion of the associated proline-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 does not harbor a transcriptional transactivation domain. Because vertebrate origins of replication have no known consensus sequence, we suggest that sequence-specific DNA binding proteins such as RIP60 may act as accessory factors in origin identification prior to the assembly of pre-initiation complexes.