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Probing ATR Activation with Model DNA Templates

The ATR kinase is a critical upstream component of a checkpoint pathway that responds to many forms of damaged and incompletely replicated DNA. Cellular processes such as DNA replication and repair are thought to convert these DNA lesions into a common DNA intermediate that activates this signaling pathway. Indeed, numerous studies have shown that two DNA structures formed during these processes – single-stranded DNA (ssDNA) and junctions between double-stranded DNA (dsDNA) and ssDNA – are important components of the ATR-activating structure. However, an unanswered question is whether primed ssDNA is sufficient for activation of the ATR response. We recently demonstrated that primed ssDNA is sufficient to induce a bona fide checkpoint response in Xenopus egg extracts. This is the first well-defined DNA structure capable of eliciting ATR activation. Using this structure, we examined the contribution of ds/ssDNA junctions and ssDNA to checkpoint activation. Our results indicate the context in which the checkpoint-activating structure is generated may contribute significantly to its signaling properties. Here we discuss the implications of our findings, in the context of other recent work in the field, on our understanding of checkpoint signaling.