The Fkh1 Forkhead associated domain promotes ORC binding to a subset of DNA replication origins in budding yeast

• Published in: Nucleic acids research 2021-10, Vol.49 (18), p.10207-10220
• Main Authors: Hoggard, Timothy, Hollatz, Allison J, Cherney, Rachel E, Seman, Melissa R, Fox, Catherine A
• Format: Article
• Language: English
• Subjects: Cell Cycle Proteins - metabolism; DNA Replication; DNA, Fungal - metabolism; Forkhead Transcription Factors - metabolism; NAR Breakthrough; Origin Recognition Complex - metabolism; Protein Binding; Protein Domains; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab450

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 origins 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.