Model illustrates the key defects in H3-G34R cells and how these may contribute to genomic instability. Replication forks provide an endogenous source of DNA damage, as they are fragile structures that are prone to stalling and collapsing. (left) To cope with replication stress, cells activate the checkpoint signaling pathway by sensing ssDNA via Rad3. Rad3 interacts with ssDNA through RPA and phosphorylates H2A, which helps to transduce the checkpoint response to downstream kinases. For lesions that require HR-directed repair, nucleosome removal and subsequent 5’ to 3’ DNA resection exposes ssDNA which is initially coated with RPA, and then replaced by Rad51, facilitated by the Rad52 protein. The search for a homologous template can begin once sufficient Rad51-ssDNA filament has been formed and strand invasion of the Rad51-ssDNA filament generates Holiday junctions which need to be resolved prior to cell division. (right) In H3-G34R cells, HR is defective. Checkpoint signaling is functional, but appears delayed with slower accumulation of γH2A possibly due to impaired nucleosome removal and delayed resection, which would limit the amount of Rad3 on RPA/DNA. Retention of γH2A indicates a defect in nucleosome removal and resection which in turn, could account for the delayed Rad52 foci formation in H3-G34R cells and likely delayed Holiday junction resolution following repair. H3-G34R cells show elevated chromosome segregation defects without defects in heterochromatin or kinetochore chromatin, suggestive that a delayed resolution of HR products contributes to genomic instability.