Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions [ChIP-seq]

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-phase of the cell cycle. Unscheduled G1 replication initiated at canonical S-phase origins across the genome. We quantified differences in replisomes in G1- and S-phase and identified firing factors, polymerase a, and histone supply as factors that limit replication outside S-phase. G1 replication per se did not trigger cellular checkpoints. Subsequent replication during S-phase, however, resulted in over-replication and led to chromosome breaks via head-to-tail replication fork collisions that are marked by chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA. Low-level, sporadic induction of G1 replication induced an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation by G1/S deregulation. Overall design: ChIP-seq of EdU-labeled DNA synthesized in G1-arrested wild-type S. cerevisiae cells or cells undergoing unscheduled replication in G1 through bypassing CDK and DDK control of replication initiation. Strand-specific ChIP-seq of RPA-bound DNA at different timepoints in wild-type and rad52? cells after unscheduled replication in G1 and in cells undergoing sporadic G1 replication through BiFC-mediated stabilization of the interaction between replication initiation proteins Dpb11 and Sld2. n=2 replicates for each experiment.