The Fkh1 Forkhead Associated Domain Promotes ORC Binding to a Subset of DNA Replication Origins in Budding Yeast

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5 of and proximal to their ORC sites (5-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5 FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins requirement for their 5 FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.