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.
Chondrocyte hypertrophic differentiation, a key process in endochondral ossification (EO), is also a feature of osteoarthritis leading to articular cartilage destruction. ShcA (Src homology and Collagen A) is an adaptor protein that binds to the cytoplasmic tail of receptor tyrosine kinases. We found that deletion of ShcA in chondrocytes of mice inhibits hypertrophic differentiation, alters the EO process, and leads to dwarfism. ShcA promotes ERK1/2 activation, nuclear translocation of the master transcription factor for chondrocyte hypertrophy, RunX2, while maintaining the Runx2 inhibitor YAP1 in its cytosolic inactive form. This leads to hypertrophic commitment and expression of markers of hypertrophy, such as Collagen X. In addition, ShcA deletion in chondrocytes protects from age-related osteoarthritis development in mice. Our results reveal that ShcA integrates multiple stimuli which affect the intracellular signaling processes leading to the hypertrophic commitment of chondrocytes and osteoarthritis.
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