ATR signaling at a glance

The maintenance of genomic integrity is crucial for the survival of all organisms. In humans, compromised genomic integrity contributes to genetic disorders, aging and cancers. The task of safeguarding the genome is accomplished by the concerted action of a number of cellular processes, including DNA replication, DNA repair, senescence(definition) and apoptosis. Many, if not all, of these processes are regulated and coordinated by the DNA-damage checkpoint, which is a complex signaling network that is triggered by DNA damage or genomic instability. Two phospho-inositide 3-kinase-like protein kinases (PIKKs) – ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) – are master regulators of two major checkpoint pathways (Harper and Elledge, 2007). ATM is primarily activated by DNA double-strand breaks (DSBs), whereas ATR responds to a much broader spectrum of DNA damage, including DSBs and many types of DNA damage that interfere with DNA replication (Cimprich and Cortez, 2008; Zou, 2007). In contrast to ATM, ATR has a crucial role in stabilizing the genome during DNA replication and is essential for cell survival (Brown and Baltimore, 2003). Here, we summarize the recent findings on ATR signaling in four areas: sensing of DNA damage; activation of the ATR kinase; interplay between ATR and ATM; and phosphorylation of ATR substrates.The initial step in ATR activation is the recognition of DNA structures that are induced by DNA damage, such as single-stranded DNA (ssDNA) and junctions between ssDNA and double-stranded DNA (dsDNA). ssDNA is a DNA structure that is commonly induced by DNA damage and by interference with DNA replication. An increased amount of ssDNA is generated at DNA replication forks when the coordination between DNA polymerase activity and DNA helicase activity is compromised (Byun et al., 2005; Walter and Newport, 2000). In addition, ssDNA gaps are induced by several types of DNA repair, such as nucleotide excision repair, mismatch repair and long-patch base excision repair (Friedberg, 2003; Lopes et al., 2006). ssDNA is also present at DSBs that have been trimmed by exo- or endonucleases through a process called resection (Lee et al., 1998). When ssDNA is generated at sites of DNA damage or at stalled DNA replication forks, it is always accompanied by junctions between ssDNA and dsDNA. Circular ssDNA that is annealed with primers, which possesses both ssDNA and ssDNA-dsDNA junctions, is sufficient to activate the ATR-mediated DNA-damage checkpoint response in Xenopus laevis egg extracts (MacDougall et al., 2007).In all eukaryotes, ssDNA that is induced by DNA damage is first detected by replication protein A (RPA), which is an ssDNA-binding protein complex. ATR-interacting protein (ATRIP), which is the regulatory partner of ATR, binds directly to RPA-coated ssDNA (RPA-ssDNA) and thereby enables the ATR-ATRIP complex to localize to sites of DNA damage (Ball et al., 2005; Namiki and Zou, 2006; Zou and Elledge, 2003). The N-terminal RPA-interacting domains of human ATRIP and its Saccharomyces cerevisiae homolog Ddc2 are important for the recruitment of ATRIP and Ddc2 to sites of DNA damage (Ball et al., 2007; Ball et al., 2005; Zou and Elledge, 2003). Additional interactions between ATR-ATRIP and RPA, as well as the interactions between ATR-ATRIP and other proteins, might also contribute to the association of ATR-ATRIP with damaged DNA (Ball et al., 2005; Namiki and Zou, 2006; Yoshioka et al., 2006). The localization of ATR-ATRIP to sites of DNA damage, however, is by itself not sufficient to activate the DNA-damage-checkpoint response fully; instead, it requires the functions of additional ATR regulators, including RAD17, RAD9, RAD1 and HUS1. RAD17 is a protein that is structurally related to RFC1 (the largest subunit of the five-subunit replication complex RFC), and it forms an RFC-like complex with the four small subunits of RFC (RFC2-RFC5). By contrast, RAD9, RAD1 and HUS1 form a ring-shaped trimeric complex (the 9-1-1 complex) that resembles proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerase. During DNA replication, RFC recognizes primer-template junctions and recruits PCNA onto DNA; similarly, during the DNA-damage response, the RAD17 complex recruits 9-1-1 complexes onto damaged DNA. Both RFC and the RAD17 complex require RPA for their function (Ellison and Stillman, 2003; Majka et al., 2006a; Zou et al., 2003). Unlike RFC, which specifically recognizes 3′ dsDNA-ssDNA junctions, the RAD17 complex preferentially recruits 9-1-1 complexes to 5′ dsDNA-ssDNA junctions, which are associated with resected DSBs and stressed replication forks (Ellison and Stillman, 2003; Majka et al., 2006a; Zou et al., 2003). The recognition of DNA damage by ATR-ATRIP and the RAD17 complex is largely independent (Kondo et al., 2001; Melo et al., 2001; Zou et al., 2002); in S. cerevisiae, colocalization of Ddc2 (which is homologous to ATRIP) and Ddc1 (which is homologous to RAD9) at artificial binding sites activates the DNA-damage checkpoint in the absence of DNA damage (Bonilla et al., 2008), suggesting that a crucial function of DNA damage in checkpoint activation is to bring ATR-ATRIP and the 9-1-1 complex together.Following the recruitment of ATR-ATRIP and the 9-1-1 complex, the kinase activity of ATR is stimulated at sites of DNA damage. When it is loaded onto DNA, the S. cerevisiae Ddc1-Rad17-Mec3 complex (which contains homologs of the three components of the 9-1-1 complex) directly stimulates the kinase activity of the Mec1-Ddc2 complex (which contains homologs of ATR and ATRIP) (Majka et al., 2006b). Whether the 9-1-1 complex can stimulate ATR-ATRIP activity in other organisms is still unclear. In both human and Xenopus, DNA topoisomerase II binding protein 1 (TopBP1) stimulates the kinase activity of ATR-ATRIP even in the absence of DNA (Kumagai et al., 2006). Dpb11, the S. cerevisiae homolog of TopBP1, also stimulates the kinase activity of Mec1-D