Engineering cellular systems to study replication stress

Abstract

The process of DNA replication is unique in cell biology since all errors will irreversibly change the nature of the cellular progeny. Failure during DNA replication – so-called DNA replication stress has been shown to be critical for genome stability and a key driver of cancer. A key limitation in our understanding of replication stress comes from the fact that it is a poorly defined phenomenon that encompasses several distinct molecular mechanisms, including replication-transcription collisions, over- and under-replication and replication stalling. Currently, we lack a molecular marker to differentiate between different forms of replication stress and utilize this knowledge in cancer therapy. Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1- phase of the cell cycle. We characterize the mechanism of G1 replication as well as factors that restrain DNA replication outside of S phase. G1 replication is highly toxic and induces DNA damage in subsequent cell cycle phases. We show that - mechanistically - subsequent replication during S-phase results in over-replication and chromosome breaks via replication collisions. Notably, single-stranded DNA accumulate with a unique chromosome-wide and strand-biased pattern, which allowed to deduce a mechanism of head-to-tail fork collisions upon over-replication. The signature of ssDNA accumulation is therefore an excellent marker for replication stress by unscheduled replication. The utilization of ssDNA signatures to discriminate different forms of replication stress, for example after replication-transcription conflicts and accumulation of RNA-DNA hybrids will be discussed.