Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.

Replication of a eukaryotic genome is a challenging task that is often made more difficult by conditions that interfere with replisome progression. These circumstances include DNA lesions that obstruct replicative polymerases, drugs that target DNA polymerases or enzymes required to synthesize deoxynucleoside triphosphates (dNTPs), and protein complexes bound to DNA (7, 36). Stalled replication forks are prone to collapse, regression, and recombination (32). Collapsed forks are among the most serious forms of DNA damage and as such pose a grave threat to cell survival and genome integrity (28). Discovering how cells cope with aberrant replication forks is therefore essential for understanding mechanisms of genome maintenance. Studies of budding and fission yeasts have uncovered a network of proteins that form the replication checkpoint (7, 36). Central to this network are protein kinases of the ATM/ATR family, such as Mec1 in the budding yeast Saccharomyces cerevisiae and Rad3 in the fission yeast Schizosaccharomyces pombe. In fission yeast, Rad3 forms a complex with Rad26 and functions together with a trimeric checkpoint clamp (Rad1-Rad9-Hus1) and five-subunit checkpoint clamp loader (Rad17-RFC2-RFC3-RFC4-RFC5) to sense stalled replication forks and transmit a checkpoint signal. These proteins act together with Mrc1, a mediator of the replication checkpoint, to activate the replication checkpoint effector Cds1, a protein kinase homologous to Rad53 in budding yeast and Chk2 in humans (1, 46). Budding yeast rad53 mutants starved of dNTPs will arrest in S phase and accumulate aberrant DNA structures such as regressed forks or hemireplicated regions, and they are unable to fully resolve replication intermediates when dNTP levels are restored (30, 44, 47). How Rad53 preserves stalled forks is unknown, but there is accumulating evidence that it and Cds1 control phosphorylation of several replication and recombination proteins (7, 36). These proteins include Mus81, a subunit of the Mus81-Eme1 DNA endonuclease complex. Mus81-Eme1 is required for recovery from collapsed forks and is thought to be a component of Holliday junction resolvase in fission yeast. Mus81 physically interacts with Cds1 homologs in budding yeast, fission yeast, and humans (5, 6, 11, 22). Mammalian Chk2 plays an important role in controlling the apoptotic response to DNA damage, apparently through its ability to control the p53 tumor suppressor, and recent evidence has indicated that Chk2 is a bona fide tumor suppressor (21, 45). In some situations, stalled forks are important for cell vitality, as is the case for cell type switching in fission yeast (12, 13). Mating type in fission yeast is determined by the expressed gene cassette at the mat1 locus. Mating type switching occurs when the DNA cassette at mat1 is replaced by one of two silent donor cassettes. Switching requires a strand-specific imprinting event that occurs when mat1 is replicated in a specific direction. The direction of replication of mat1 is determined by polar replication fork pausing and termination sites located near mat1 and is dependent on several proteins. One of these proteins is Swi1, a 971-amino acid (aa) protein that has ∼25% sequence identity to Drosophila melanogaster Timeless, mammalian Tim1, and budding yeast Tof1 (13). Timeless controls circadian rhythms in Drosophila (35), whereas mouse Tim1 is an essential nuclear protein that does not regulate circadian rhythms but whose function is unknown (19). Tof1, first identified as topoisomerase 1-associated factor in a two-hybrid screen, is involved in DNA damage responses during S phase (16). Mutant tof1 cells exhibit no obvious phenotypes and are not sensitive to genotoxic agents, but deletion of TOF1 enhances the genotoxic-sensitive phenotype of rad9 mutants. Rad9 is a mediator protein that facilitates activation of Rad53 by Mec1 (18, 42). Deletion of TOF1 exacerbated the Rad53 activation defect of rad9 mutants, suggesting that Tof1 and Rad9 act in redundant pathways to control Rad53 activation (16). Fission yeast swi1 mutants have additional phenotypes that are unconnected to mating type switching (13). They have a reduced growth rate that is exacerbated by mutations in top1, which encodes topoisomerase I. Swi1 is essential for viability in a mutant that is partially defective for DNA polymerase alpha. These studies indicated that Swi1 might have a more general role in DNA replication. Here we report that Swi1 is crucial for survival of replication fork arrest. Swi1 has both Cds1-dependent and -independent functions. Swi1 appears to act early in the response to fork arrest, perhaps as a replisome component, to stabilize forks in a configuration that is recognized by the replication checkpoint sensors.