The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of

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... igins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5′ of and proximal to their ORC sites (5′-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5′ FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5′ FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at ∼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding.