Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions

Abstract

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 engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads 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 induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

“…2a ). As an alternative system to interfere with DDK function, we generated an auxin-inducible degron (AID) strain 69 , 70 , which allowed conditional depletion of Dbf4-3AID (specifically, three copies of miniAID tag) by addition of Indole-3-acetic acid (IAA) or 1-Naphthaleneacetic acid (NAA) to the growth medium (Supplementary Fig. 2b, c ).…”

Large tandem duplications in cancer result from transcription and DNA replication collisions

“…1a ). Such dosage imbalance will lead to strand bias 39 of the resulting SVs. The SV type depends on the choice of repair partner.…”

A R-loop sensing pathway mediates the relocation of transcribed genes to nuclear pore complexes

“…We therefore directed our attention to RPA, the main cellular ssDNA-binding complex, which was previously localized to transcribed genes in yeast and associated with R-loop sensing and resolution in mammalian cells 44 , 61 . To specifically assess the presence of RPA at R-loop forming genes without the confounding effect of its replication-dependent recruitment, we used a strand-specific RPA ChIP-seq dataset obtained from non-cycling yeast cells 62 . By restricting our analysis of RPA occupancy to intronless and intron-containing highly transcribed genes (same groups as Fig.…”

“…The following calculations were used to evaluate biases in base content: GC skew = (G-C)/(G + C); AT skew = (A-T)/(A + T). Nic96 (this study), RPA 62 and R-loop 38 occupancies were determined by integrating ChIP- or DRIP-seq counts over transcription units (Fig. 1d ; Supplementary Figs.…”