Cells with reduced origin firing have an increased rate of replication fork progression, whereas fork progression is slowed in cells with excess origins.

Forkhead Box (Fox) proteins share the Forkhead domain, a wingedhelix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G 1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1-and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.DNA replication timing | chromatin | nuclear organization | Fox proteins | DNA binding protein F undamental processes of DNA repair, recombination, transcription, and replication often occur in specific subnuclear domains or in localized foci (reviewed in refs. 1-4). In yeast for example, hundreds of highly expressed tRNA genes coalesce into multiple foci, each containing several active tRNA genes (reviewed in ref. 5). Similarly, hundreds of replication origins coalesce into foci containing several origins each, which become bidirectional replisomes that remain colocalized as DNA is spooled through during replication (reviewed in ref. 6). Such spatial organization is thought to contribute to the efficiency of these processes by increasing the local concentration of the involved factors, which may consequently also exclude competing or interfering factors or processes. How distal DNA sequences are assembled into these structures is poorly understood.We recently identified the Saccharomyces cerevisiae transcription factors Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as being required for the clustering of a subset of replication origins in G 1 phase and for the early initiation of these origins in the ensuing S phase (7). How Fkh1 and Fkh2 promote clustering is unclear; however, their binding near origins might promote origin-origin interactions through binding to other proteins at origins, such as the origin recognition complex (ORC) (7). Fkh1 has also been implicated as a regulator of mating-type switching, which involves homologous recombination between distal chr...

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.

The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.