ATR:ATRIP complex is recruited to resected DNA double-strand breaks (DSBs) via interaction with the RPA complex that coats single strand DNA (ssDNA) 3'-overhangs, but this is not sufficient for ATR to become catalytically active. ATR kinase activity requires the presence of the RAD17:RFC complex, RAD9:HUS1:RAD1 (9-1-1) complex and TOPBP1. RAD17:RFC loads RAD9:HUS1:RAD1 onto junctions of single strand and double-strand DNA (ssDNA-dsDNA junctions), present at resected DNA DSBs (Bermudez et al. 2003, reviewed by Sancar et al. 2004). TOPBP1 binds the C-terminal tail of RAD9, and is thus brought in the proximity of ATR, where it can activate it (Kumagai et al. 2006, Delacroix et al. 2007). The interaction of TOPBP1 and RBBP8 (CtIP) also contributes to TOPBP1 loading (Ramirez-Lugo et al. 2011). Phosphorylation of ATR at threonine residue T1989 may create a binding site for the BRCT domains of TOPBP1 (Liu et al. 2011). It is not clear whether T1989 of ATR is phosphorylated through autophosphorylation (Liu et al. 2011), as it does not conform to the SQ/TQ consensus, or by another kinase (Liu et al. 2013). RHNO1 (RHINO) protein simultaneously binds RAD9:HUS1:RAD1 complex and TOPBP1 and is required for the full catalytic activation of ATR (Cotta-Ramusino et al. 2011).

CHEK1 (Chk1) is a checkpoint kinase activated during genotoxic stress. CHEK1 activation at resected DNA double-strand breaks (DSBs) involves ATR-mediated phosphorylation of CHEK1 serine residues S317 and S345 in the presence of claspin (CLSPN), TOPBP1, RAD17:RFC complex, RAD9:HUS1:RAD1 complex, TIMELESS:TIPIN complex, RPA complex and RHNO1 (Liu et al. 2000, Zhao and Piwnica-Worms 2001, Kumagai and Dunphy 2003, Sorensen et al. 2004, Wang et al. 2006, Kemp et al. 2010, Cotta-Ramusino et al. 2011, Liu et al. 2012). Following phosphorylation, CHEK1 dissociates from chromatin and phosphorylates target proteins involved in S/G2 checkpoint activation and/or homologous recombination repair (Smits et al. 2006). CLSPN needs to interact with chromatin only transiently in order to facilitate CHEK1 activation (Lee et al. 2005).

Once the Mcm2-7 complex has been assembled onto the origin of replication, the next step is the assembly of Cdc45, an essential replication protein, in late G1. The assembly of Cdc45 onto origins of replication forms a complex distinct from the pre-replicative complex, sometimes called the pre-initiation complex. The assembly of Cdc45 onto origins correlates with the time of initiation. Like the Mcm2-7 proteins, Cdc45 binds specifically to origins in the G1 phase of the cell cycle and then to non-origin DNA during S phase and is therefore thought to travel with the replication fork. Indeed, S. cerevisiae Cdc45 is required for DNA replication elongation as well as replication initiation. Cdc45 is required for the association of alpha DNA polymerase:primase with chromatin. Based on this observation and the observation that in S. cerevisiae, cCdc45 has been found in large complexes with some components of Mcm2-7 complex, it has been suggested that Cdc45 plays a scaffolding role at the replication fork, coupling Pol-alpha:primase to the replication fork through the helicase. Association of Cdc45 with origin DNA is regulated in the cell cycle and its association is dependent on the activity of cyclin-dependent kinases but not the Cdc7/Dbf4 kinase. In Xenopus egg extracts, association of Cdc45 with chromatin is dependent on Xmus101. TopBP1, the human homolog of Xmus1, is essential for DNA replication and interacts with DNA polymerase epsilon, one of the polymerases involved in replicating the genome. TopBP1 homologs have been found in S. cerevisiae and S. pombe. Sld3, an additional protein required for Cdc45 association with chromatin in S. cerevisiae and S. pombe, has no known human homolog.

ATR phosphorylates several proteins at DNA insterstrand crosslinks (ICL-DNA), with ATR activity at ICL-DNA being independent of the presence of RAD17 and TOPBP1 (Shigechi et al. 2012, Tomida et al. 2013). Besides phosphorylating the RPA2 subunit of the RPA heterotrimeric complex (Huang et al. 2010), activated ATR also phosphorylates the Fanconi anemia core complex component FANCM on serine residue S1045 (Singh et al. 2013). ATR-mediated phosphorylation of FANCM is thought to be important for the progression of ICL repair, although the mechanism is not known. The critical ATR substrate at ICL-DNA is considered to be FANCI component of the ID2 complex. ATR-mediated phosphorylation of FANCI, at least on serine residues S556, S559, S565 and S617, is a prerequisite for FANCD2 monoubiquitination (Ishiai et al. 2008, Shigechi et al. 2012). FANDC2 itself is also phosphorylated by ATR on threonine residue T691 and serine residue S717, which promotes FANCD2 monoubiquitination and enhances cellular resistance to DNA crosslinking agents (Ho et al. 2006).

Homology directed repair (HDR) through homologous recombination (HRR) or single strand annealing (SSA) requires extensive resection of DNA double-strand break (DSB) ends (Thompson and Limoli 2003, Ciccia and Elledge 2010). The resection is performed in a two-step process, where the MRN complex (MRE11A:RAD50:NBN) and RBBP8 (CtIP) bound to BRCA1 initiate the resection. This step is regulated by the complex of CDK2 and CCNA (cyclin A), ensuring the initiation of HRR during S and G2 phases of the cell cycle, when sister chromatids are available. The initial resection is also regulated by ATM-mediated phosphorylation of RBBP8 and CHEK2-mediated phosphorylation of BRCA1 (Chen et al. 2008, Yun and Hiom 2009, Buis et al. 2012, Wang et al. 2013, Davies et al. 2015, Parameswaran et al. 2015). After the initial resection, DNA nucleases EXO1 and/or DNA2 perform long-range resection, which is facilitated by DNA helicases BLM or WRN, as well as BRIP1 (BACH1) (Chen et al. 2008, Nimonkar et al. 2011, Sturzenegger et al. 2014, Suhasini et al. 2011). The resulting long 3'-ssDNA overhangs are coated by the RPA heterotrimers (RPA1:RPA2:RPA3), which recruit ATR:ATRIP complexes to DNA DSBs and, in collaboration with RAD17:RFC and RAD9:HUS1:RAD1 complexes, and TOPBP1 and RHNO1, activate ATR signaling. Activated ATR phosphorylates RPA2 and activates CHEK1 (Cotta-Ramusino et al. 2011), both of which are necessary prerequisites for the subsequent steps in HRR and SSA.