SRA

In Saccharomyces cerevisiae the forkhead (Fkh) transcription factor Fkh1 forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Fkh1 binds directly to origin-adjacent Fkh1 binding sites (FKH sites), providing evidence that Fkh1 acts directly. However, the post-DNA binding functions Fkh1 uses to promote early origin activity are not defined. Fkh1 contains a conserved FHA (fork head associated) domain, a protein-binding module that binds to phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex) -origin binding step, the G1-phase event that initiates the origin cycle. The relevance of the Fkh1-FHA to chromosomal replication or ORC-origin interactions at genome scale has not been reported. Here, S-phase SortSeq experiments were used to assess genome replication in proliferating FKH1 and fkh1R80A mutant cells. The data provided evidence that the Fkh1-FHA domain promoted the activity of ~ 100 origins that act in early S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ~ 100 late origins. ORC ChIPSeq data provided evidence that the Fkh1-FHA domain promoted normal ORC-origin binding at FHA-stimulated origins. Origins are associated with a distinctive nucleosome-organization that frames a nucleosome deplected region (NDR) over the origin, and ORC contributes to this architecture. To ask whether the Fkh1-FHA domain had an impact on the ORC- and nucleosome protein-DNA interactions at origins, MNaseSeq data were generated from G1-arrested and proliferating FKH1 and fkh1R80A mutant cell populations. These data provided evidence that origin groups differentially regulated by the Fkh1-FHA domain were characterized by distinct G1-phase ORC- and nucleosome configurations that were Fkh1-FHA domain-dependent. Thus, the Fkh1-FHA domain performed an important post-DNA binding role of the Fkh1 protein in promoting hallmarks of G1-phase origin-associated protein-DNA architecture and the activity of early origins.