title: Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex.
abstract: 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.
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Allan
authors.initials: A
authors.lastname: Reinapae
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Jalakas
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Nikita
authors.initials: N
authors.lastname: Avvakumov
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Marko
authors.initials: M
authors.lastname: Lõoke
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Kersti
authors.initials: K
authors.lastname: Kristjuhan
authors.affiliation: Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Arnold
authors.identifier: 0000-0002-5842-4952
authors.initials: A
authors.lastname: Kristjuhan
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C464906:Fkh1 protein, S cerevisiae; D051858:Forkhead Transcription Factors; D029701:Saccharomyces cerevisiae Proteins; C069199:MCM2 protein, S cerevisiae