title: Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.
abstract: The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.
authors.affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: Jared M
authors.initials: JM
authors.lastname: Peace
authors.affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: Sandra K
authors.initials: SK
authors.lastname: Villwock
authors.affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: John L
authors.initials: JL
authors.lastname: Zeytounian
authors.affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Aparicio
mesh_terms: D001973:Bromodeoxyuridine; D018797:Cell Cycle Proteins / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004261:DNA Replication; D042522:DNA Replication Timing; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D016193:G1 Phase / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C464906:Fkh1 protein, S cerevisiae; C464958:Fkh2 protein, S cerevisiae; D051858:Forkhead Transcription Factors; D029701:Saccharomyces cerevisiae Proteins; D001973:Bromodeoxyuridine