DNA damage checkpoint in S phase is the replication initiation factor Cdc45, which upon DNA damage is prevented from being loaded at DNA replication origins in an ATR- and Chk1-dependent manner. An essential protein for ATR kinase activation is the ATRinteracting protein ATRIP, which is constitutively bound to ATR and facilitates the recruitment of ATR to DNA. However, there are several proposed mechanisms by which the ATR/ ATRIP complex and the ATR-Chk1 pathway may become activated by genotoxic stress. Though the ATR/ATRIP complex may directly sense DNA damage itself or through association with its activator protein TopBP1, a variety of other protein factors are also implicated in the direct recognition of DNA damage and replication stress. These DNA damage “sensor” proteins include a variety of DNA repair factors that directly associate with specific forms of DNA damage, such as bulky DNA adducts, DNA mismatches, interstrand crosslinks, single-stranded DNA, and primer-template junctions. Through additional protein-protein interactions, these repair factors may directly and stably recruit the ATR kinase to the DNA damage site to initiate signaling responses. Two of the most prominent ATR-mediated DNA damage checkpoint “sensor” proteins include Replication Protein A, a ssDNA-binding protein that binds the ATR-interacting protein ATRIP to recruit the ATR kinase to sites of DNA damage, and the primer-template junction clamp complex Rad9-Hus1- Rad1, which through a direct protein-protein interaction brings TopBP1 into proximity of ATR to enable full activation of ATR kinase GSI-IX activity. There are also additional factors that may aid the recruitment or activation of ATR at specific forms of DNA base damage, such as the nucleotide excision repair factor XPA, the Fanconi Anemia-associated factor FAAP24, and the mismatch repair protein MSH2. An additional class of protein factors has been suggested to facilitate the specific phosphorylation of Chk1 or other substrates by ATR in order to amplify or maintain checkpoint signaling responses. These checkpoint “mediator” proteins include the direct ATR kinase-activating protein TopBP1 and the Chk1- interacting factor Claspin. Similarly, based on the ability of the Tipin subunit to directly bind both RPA and Claspin, the Timeless-Tipin complex may mediate Chk1 phosphorylation by ATR at sites of DNA damage and replication stress bound by RPA. Though a great deal of progress has been made in significant questions remain regarding the DNA substrates and protein-DNA interactions that trigger utilization.