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. 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.

癌症中，遏制DNA过度复制的调控机制常发生失调，诱导癌细胞进入过度复制状态是一种颇具前景的治疗策略。我们对因调控互作紊乱而发生部分基因组再复制的癌细胞（下称“再复制细胞”）的DNA合成模式展开了系统性解析。此类细胞呈现出复制速率放缓、复制起始事件频率升高，以及一种优先激活早期复制起始位点的偏斜起始模式。与暴露于复制应激的细胞不同，后者会激活一类此前未被利用的全新“休眠”复制起始位点；而本研究中优先激活的再复制起始位点，均源自与正常生长状态下激活的起始位点相同的潜在起始位点库。从机制层面来看，这种偏斜的起始模式，源于复制起始前，预复制复合物（pre-replication complexes）在已许可染色质的不同区域分布不均衡。这种独特的模式表明，打破正常情况下抑制DNA过度复制的强调控互作，至少可通过两条通路实现，每条通路各自激活一套特定的复制起始位点。研究人员采用λ核酸外切酶（lambda exonuclease）法，从HCT116细胞与U2OS细胞中纯化新生DNA链；通过溴脱氧尿苷（BrdU）氯化铯梯度离心法分离得到再复制DNA；对HCT116细胞的染色质开展针对CDT1与pS139-MCM2抗体的染色质免疫共沉淀测序（ChIP-Seq）。