These results are consistent with the purported role for these proteins as a “replication fork-protection complex” that stabilizes replication forks that stall due to DNA damage or abnormal DNA structures. Similarly, Claspin and its yeast homolog Mrc1 were previously shown to preferentially associate with branched DNA structures by electrophoretic mobility shift assay and electron microscopy. The preferential association of both the Timeless-Tipin complex and its individual subunits with the branched DNA indicates that several proteinDNA interactions may be involved in the binding of the complex to DNA. Though the relative amount of Tipin that associated with the DNA was less than that for Claspin or Timeless, at its highest DAPT 208255-80-5 concentration in the binding reaction, it showed a much greater preference for the branched DNA in comparison to either the ssDNA or dsDNA. These results suggest that Tipin may play an important role in detecting branched DNA structures. In contrast to the proteins just discussed, TopBP1 did not show any preference for any particular DNA structure under these reaction conditions, indicating that it cannot discriminate between these different forms of DNA under low-stringency conditions. Cdc45, a target of the ATR-Chk1 intra-S phase DNA damage checkpoint response, did not associate with any DNA structure under a variety of conditions tested. Interestingly, as shown in Figure 8, both Claspin and Tipin showed a stronger association with the AAFdamaged DNA than the undamaged DNA. Analysis of fragments of these proteins identified smaller domains that are sufficient for this characteristic binding property. Though both of these factors mediate Chk1 phosphorylation by ATR in response to UV and UV-mimetic agents, there has previously been no evidence that these proteins directly recognize bulky DNA adducts. However, since both Claspin and Tipin showed increased affinity for the branched DNA structure in comparison to either ssDNA or dsDNA, these results indicate that the recognition of multiple checkpoint-inducing DNA structures by these proteins may contribute to their DNA damage checkpoint functions. The diversity and number of protein-DNA interactions that are involved in activating the ATR-Chk1 pathway in response to DNA damage and replication stress remain unclear. Based on a variety of genetic, biochemical, and cell biological approaches, strong evidence supports the notion that ssDNA and primertemplate junctions are two primary components of ATR activation. Through recruitment of ATR-ATRIP to ssDNA by RPA.