DYNAMICS OF REPLICATION ORIGIN OVER-ACTIVATION

Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions ("re-replicating cells"). These cells exhibited slow replication, increased frequency of replication initiation events and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (”dormant”) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins. Overall design: Nascent strands were purified with the lambda exonuclease methods from HCT116 cells or U2OS. Re-replicated DNA was isolated by BrdU cesium chloride gradient. Chromatin from HCT116 cells was subjected to ChIP-Seq with antibody directed against CDT1 and pS139-MCM2.