* indicates a non-specific band. Sld3. This allows CDK to remain active during S phase in the presence of DNA damage, which is crucial to prevent reloading of Mcm2-7 onto origins that have already fired6. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the intra-S checkpoint is primarily due to the inhibition of origin firing. Activation of the DNA damage checkpoint kinases in S-phase regulates genomic replication in at least two ways: firstly by protecting stalled replication forks11-14 and secondly by blocking further origin firing7-10. To determine whether the DNA replication machinery is directly regulated by checkpoints, we set out to identify Rad53 substrates in the budding yeast, Rad53 kinase assay with bacterially expressed Sld3 fragments 1-5. E) Western blots of purified and cleaved Sld3-TEV allele from HU arrested cells. This allele contains a myc tag at the C-terminus, HA-tag in the middle, with a TEV protease cleavage site in-between. This allele is viable as the only copy in yeast. F) Western blot of Sld3-13myc from cells arrested in G1 with alpha factor and released into HU for the indicated times. The magnitude of the shift and the multitude of bands seen in SDS-PAGE (Figure 1b) indicated that the serine/threonine-rich Sld3 protein (Figure 1c) is multiply phosphorylated after checkpoint activation. We used purified Rad53 to phosphorylate a series of Sld3 fragments (Figure 1c) we phosphorylated SB366791 arrays of peptides corresponding to the entire Sld3 amino acid sequence attached to a cellulose membrane. Consistent with Figure 1d, most of the phosphorylated peptides occurred within the C-terminal domain of IL10A Sld3 (Supp. Figure 1c). Because of the extensive overlap in the peptides on the array (Supp. Figure 2a-d) most sites could be identified unambiguously. All 38 potential serine and threonine phosphorylation sites were mutated to alanine (Figure 1c and Supp. Table 2). Compared to the wild type protein, this allele of Sld3 (mutants containing subsets of the 38 sites mutated to alanine all show reduced phosphorylation shift SB366791 indicating that many or most of the sites contribute to the full phosphorylation shift and Sld3 inhibition (Supp. Figure 2e,f). The residual shift in may be due to additional sites missed in our analysis or may due to be cryptic sites only phosphorylated when the stronger sites in the wild type protein are absent. Yeast strains expressing as the sole copy of Sld3 showed no sensitivity to HU or DNA damaging agents and did not exhibit synthetic growth defects with several conditional alleles of essential replication proteins (Supp. Figure 3) arguing that the Sld3-A protein is functional for DNA replication. These Rad53 sites are primarily in the C-terminal portion of Sld3, where the essential CDK sites (Thr600, Ser622) are found (Figure 1c). Figure 2a shows that, whilst CDK phosphorylation of the C-terminus of Sld3 allows direct binding to Dpb11 but not to a Dpb11 truncation lacking the first BRCT repeat (N); however, subsequent Rad53 phosphorylation of Sld3 inhibits interaction with Dpb11. Mutation of the strongest Rad53 sites in the C-terminus of Sld3 to aspartate residues (C Figure 1c, Supp. Table 2), to mimic constitutive phosphorylation, also blocks interaction with Dpb11 (Figure 2b) without blocking CDK phosphorylation (Supp. Figure 4) and is unable to support growth (Figure 2c). The CDK-dependent interaction between Sld3 and Dpb11 can be bypassed by direct covalent fusion of these proteins3. Figure 2c shows that fusion of the Sld3-12D mutant protein to Dpb11 restored its ability to support growth. This argues that Sld3 phosphorylation by Rad53 inhibits its ability to interact with Dpb11 and background also produced a protein (Sld3-14D C Figure 1c) that could not interact with SB366791 Dpb11 (Figure 2b) and could not support growth (Figure 2c). In contrast to could not support growth after fusion to Dpb11 (Figure 2c). Previous work has shown that Sld3 also interacts with Cdc45 and the GINS subunit Psf1 in a two hybrid assay16. A mutant protein in which Ser306 and Ser310 were changed to aspartate (Sld3-2D) interacted with both Dpb11 and Psf1 in a two hybrid assay (Figure 2d). However, compared to wild type, Sld3-2D showed a reduced interaction with Cdc45. Consistent with this weakened interaction, over-expression of Cdc45 allowed the Sld3-14D-Dpb11 fusion to support growth (Figure 2e). These results indicate that Sld3 phosphorylation by Rad53 inhibits interactions with both Dpb11 and Cdc45. A mutant of Sld3 in which 34 Rad53 sites.