Helicase subunit Cdc45 targets the checkpoint kinase Rad53 to both replication initiation and elongation complexes after fork stalling

Across eukaryotes, inhibition of the progression of the DNA replication machinery causes an S-phase checkpoint response. This response regulates multiple processes, including inhibition of replication initiation and fork stabilisation. How these events are coordinated remains poorly understood. Here we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to FHA-interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. In addition to regulating replication initiation, Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally we show that a Cdc45 mutation that is orthologous to an allele found in patients with Meier-Gorlin Syndrome disrupts the functional interaction with Rad53. Together we present a single mechanism by which Rad53 targets replication initiation and elongation complexes, which may be relevant to human disease. Reads were mapped to W303 genome. All the normalisation and data processing steps done by used Deeptools. ChIP data was analysed by normalising the read counts to +/-500bp of centromeric reads. For visualization, centromeric regions were masked out. For replication profiles, a dormant replicative region was defined as the 10 kb region between two late/dormant origins of maximal base pair distance. It was assumed that there was no replication in such regions after 1h HU treatment. 10 such origin pairs were chosen as the dormant region set. Replication profiles were obtained using the following methodology: 1) S-phase samples were normalized to G1 samples by binning each chromosomal region to 100bps and dividing the G1 sample counts over the S-phase samples counts. 2) The average count of the dormant region set from all samples was taken as 1N. 3) The relative count of all regions in 100bp bins was calculated as a ratio of 1N. 4) Relative ratios bigger than 2.2N were masked out.