Undamaged DNA transmits and enhances DNA damage checkpoint signals in early embryos.

During embryogenesis in Xenopus laevis, the midblastula transition (MBT) occurs upon completion of the 12th cell division and marks a number of critical changes, including the introduction of gap phases into the cell cycle and the beginning of transcription. Moreover, early embryonic cell cycles lack checkpoints that halt the cell cycle in response to DNA damage, whereas the DNA damage checkpoint functions normally in post-MBT embryos (1, 10). One possible explanation is that one or more of the biochemical components required for DNA damage checkpoint signal transduction are absent in pre-MBT embryos and, thus, zygotic transcription could be necessary to produce elements of the checkpoint pathway. However, the addition of exogenous DNA with free ends mimicking DNA double-strand breaks (DSBs) induces checkpoint activation in pre-MBT embryos or egg extracts, suggesting all components of a functional checkpoint pathway exist before the MBT (6, 11, 15). In the very early embryo, this checkpoint requires both DNA DSBs and a threshold DNA-to-cytoplasm ratio to significantly affect the cell cycle (6). Importantly, even uncut plasmid DNA can facilitate responses to DNA DSBs in pre-MBT embryos (6), suggesting a previously undefined role for undamaged threshold DNA in DNA damage checkpoint signal transduction. In eukaryotic cells, ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) kinases are activated by DNA damage and initiate signal transduction pathways that lead to checkpoint activation (28). The mechanisms of DNA damage-induced ATM and ATR activation are still largely unclear. ATM activation has been attributed to DNA damage-induced ATM autophosphorylation at serine 1981 and dissociation of inactive, dimeric ATM into an active, monomeric form (2). Although autophosphorylation of ATM is widely utilized as an indicator of ATM activation, it is dispensable for murine ATM activation (26). Interestingly, although ATM is primarily activated in the vicinity of DNA DSBs, agents altering chromatin structure can activate ATM independently of DNA damage (2). Whereas ATM activation is mainly triggered by DNA DSBs, ATR is activated by single-strand DNA (ssDNA) and stalled replication forks (34). In order to activate ATR, different forms of DNA damage must be processed into a common intermediate structure: ssDNA coated with replication protein A (34). It has also been shown that TopBP1, a BRCT domain protein, is an ATR activator (16). Upon activation, ATM and ATR phosphorylate many cellular substrates critical for controlling checkpoint responses, transcription, or apoptosis, including Chk1, Chk2, H2AX, BRCA1, NBS1, SMC1, and p53 (28). Of these ATM/ATR substrates, Chk1 and Chk2 are two serine/threonine-specific kinases classified as checkpoint kinases owing to their critical roles in regulating cell cycle checkpoints. Activated Chk1/Chk2 phosphorylate and activate the tyrosine kinase Wee1 while inhibiting Cdc25 phosphatase function through phosphorylation-directed proteolysis or 14-3-3 binding (see reference 3 for a review). Lack of Cdc25 blocks the G1/S transition through accumulation of inactive phosphorylated Cdk2, and the activation of Wee1 and inhibition of Cdc25C localization or activity by 14-3-3 binding together control inhibitory phosphorylation of Tyr 15 on Cdc2/cyclin B, a critical regulator of the G2/M transition (24). The pathway responding to DNA DSBs is highly conserved through evolution. However, ATM and ATR have different roles in yeast and mammals. In Saccharomyces cerevisiae, the ATR homolog (scMec1) plays a central role in DSB-induced checkpoint signaling (22). This contrasts with the situation in mammals, where ATM is the major kinase involved in the response to DSBs and the involvement of ATR is delayed, ATM dependent, and cell cycle regulated (14, 29). A similar DNA damage checkpoint response is also present in Xenopus egg extracts, a cell-free system that has been widely used to elucidate the biochemical basis of cell cycle regulation (20). The addition of exogenous DNA damage activates both ATM and ATR in extracts, eventually resulting in the inhibition of Cdc2 and cell cycle progression (12, 33). In this study, we show that in both Xenopus embryos and egg extracts, the presence of damage-free DNA, either as uncut plasmid DNA or Xenopus sperm chromatin, greatly increases the sensitivity of DSB-induced checkpoint signaling. We demonstrate this enhancement of DSB-induced ATM-dependent signal transduction, including ATM autophosphorylation and ATM-dependent Chk1 and Chk2 phosphorylation, occurs by recruitment of activated ATM from the soluble fraction to the undamaged threshold DNA in a γ-H2AX-dependent manner.