Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions [mRNA-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. We quantified the composition of replisomes in G1- and S-phase and identified firing factors, polymerase alpha, 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 and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. 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. Overall design: RNA-seq after polyA-enrichment of wild-type cells in S-phase or cells that were induced to initiate DNA replication in G1 (expression of Dbf4, Dpb11, sld2-T84D). G1-arrested wild-type undergoing the same treatment are included as a control. n=4 replicates for each experimental condition.