Interactions between Fkh1 monomers stabilize its binding to DNA replication origins.

Eukaryotic DNA replication is initiated from multiple genomic origins, which can be broadly categorized as firing early or late in the S phase. Several factors can influence the temporal usage of origins to determine the timing of their firing. In budding yeast, the Forkhead family proteins Fkh1 and Fkh2 bind to a subset of replication origins and activate them at the beginning of the S phase. In these origins, the Fkh1/2 binding sites are arranged in a strict configuration, suggesting that Forkhead factors must bind the origins in a specific manner. To explore these binding mechanisms in more detail, we mapped the domains of Fkh1 that were required for its role in DNA replication regulation. We found that a short region of Fkh1 near its DNA binding domain was essential for the protein to bind and activate replication origins. Analysis of purified Fkh1 proteins revealed that this region mediates dimerization of Fkh1, suggesting that intramolecular contacts of Fkh1 are required for efficient binding and regulation of DNA replication origins. We also show that the Sld3-Sld7-Cdc45 complex is recruited to Forkhead-regulated origins already in the G1 phase and that Fkh1 is constantly required to keep these factors bound on origins before the onset of the S phase. Together, our results suggest that dimerization-mediated stabilization of DNA binding by Fkh1 is crucial for its ability to activate DNA replication origins.

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex.

In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins ARS305 and ARS607. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.