A checkpoint protein recruitment domain (CRD) has been identified in the N terminus of ATRIP and Ddc2. This domain binds directly to RPA70N, recruits ATR-ATRIP/Mec1-Ddc2 complexes to sites of DNA damage, and promotes ATR-dependent checkpoint signaling in response to MMS. These findings are consistent with those of Kim et al., who reported that an N-terminal domain of Xenopus
ATRIP is required for binding to RPA (25
). RPA is a modular protein, and it often makes more than one contact with its interacting partners. Indeed, Namiki and Zou identified three large regions of ATRIP that may interact with RPA-ssDNA (37
). Since no functional data were reported, additional experiments will be required to define and study the function of any other ATRIP surfaces that make direct contacts with RPA subunits. However, our data indicate that the N-terminal CRD domains of ATRIP and Ddc2 are required for the stable binding of ATRIP/Ddc2 to RPA and are necessary for retention of ATR-ATRIP/Mec1-Ddc2 at sites of DNA damage in cells.
A model of the interaction of RPA70N with a conserved ATRIP peptide within the CRD was generated using NMR data and molecular modeling from the crystal structure of a p53 peptide bound to RPA70N. The model predicts that acidic ATRIP residues (D58 and D59) make direct contacts with basic RPA70N residues (R41 and K88) in the basic cleft of the RPA70N OB fold domain. All of these amino acids are highly conserved. As predicted by this model, mutations reversing the charges on the equivalent aspartic acid residues in Ddc2 (D12K and D13K) abrogate binding to Rfa1. Interestingly, the well-characterized rfa1-t11
mutant, which is known to be replication competent but DNA-damage-response deficient, contains a single charge-reversal mutation at K45, the residue equivalent to R41 in human RPA (49
). Indeed, as our model would predict, rfa-t11
is deficient in recruiting Ddc2 to double-strand breaks (23
) and in binding Ddc2 (H. L. Ball, unpublished data). The rfa-t11
mutant is also recombination deficient, suggesting that this basic cleft in RPA70N may be a key ligand in DNA damage responses (44
). It will be interesting to determine whether other DNA damage response proteins also contain acidic helices that bind within this cleft of RPA70N. It is also noteworthy that an ATR phosphorylation site (S68) is located within the ATRIP CRD just downstream of the acidic peptide that binds to the RPA basic cleft (21
). Moreover, RPA70N appears to interact with the RPA32 N terminus when it is phosphorylated by checkpoint kinases (6
). Therefore, phosphorylation of either ATRIP or RPA may be a means to regulate the ATR-RPA interaction.
The phenotypic consequences of disrupting the ATRIP CRD-RPA70 interaction are similar in human and yeast cells. In contrast to ATRIP or Ddc2 loss of function, cells containing mutations that disrupt the CRD are only mildly sensitive to DNA-damaging agents and partially compromised in checkpoint signaling. In fact, the response to HU is nearly indistinguishable from wild-type results despite severe defects in ATR-ATRIP/Mec1-Ddc2 localization. Functions of ATRIP in addition to RPA binding are also critical for ATR signaling. These functions include oligomerization (2
), ATR stabilization (12
), and an undefined activity important for TopBP1-dependent activation of ATR.
The reason for the increased sensitivity of Ddc2 lacking the CRD to damage that generates DNA adducts (MMS) compared to depletion of nucleotides (HU) is unknown. Both types of genotoxic stress activate Mec1 during replication and stall replication forks (48
). One potential explanation for this difference may be the amount of RPA-ssDNA present at various types of DNA lesions. Mec1-Ddc2ΔN complexes may have some residual association with Rfa1 and can still partially localize to double-strand breaks. Perhaps there is more RPA-ssDNA at an HU-stalled fork than at an MMS-induced lesion, increasing the requirement for the Ddc2 CRD at the MMS lesion. Alternatively, the recruitment and activation mechanisms of ATR-ATRIP and Mec1-Ddc2 at MMS or HU lesions may be different. Accumulating evidence suggests that additional protein-protein and protein-DNA interactions other than the ATRIP-RPA interaction may help recruit ATR-ATRIP to DNA lesions (8
). Also, Ddc2 contains a DNA end-binding activity localized to a region C terminal to the predicted coiled-coil domain (42
). Perhaps these alternative modes of ATR-ATRIP/Mec1-Ddc2 recruitment function differently at HU and MMS lesions.
Consistent with the report by Kumagai and coworkers, we have found that TopBP1 activates ATR and that TopBP1-dependent activation of ATR is ATRIP dependent and does not require the ATRIP CRD (28
). RPA-ssDNA, in contrast, does not stimulate ATR kinase activity in immune complex in vitro kinase reactions, and TopBP1-dependent activation of ATR is not altered by adding RPA or RPA-ssDNA to the kinase reaction. These results suggest that TopBP1-dependent ATR activation can be separated from ATRIP-RPA binding. The affinity of TopBP1 for ATR-ATRIP is weak and difficult to detect by coimmunoprecipitations (28
). The accumulation of ATR-ATRIP and TopBP1 at sites of damage may facilitate this low-affinity interaction by increasing the local concentration of these proteins.
Taken together, these data support a multistep model proposed by Dunphy and colleagues (27
) for the activation of ATR checkpoint signaling. ATR recruitment to sites of DNA damage and replication stress occurs in part through a direct interaction between the ATRIP CRD and RPA70N. TopBP1 is recruited independently through an interaction with Rad9 (16
). The assembly of ATR-ATRIP and TopBP1 at the lesion facilitates TopBP1-dependent ATR activation and, in turn, phosphorylation of ATR substrates. Accessory proteins such as claspin are also required for phosphorylation of specific substrates (26
). Localization may also serve to bring ATR to the vicinity of key substrates involved in fork stabilization or other aspects of checkpoint regulation. Within this model, ATRIP is a key ATR regulator since it promotes both the localization and activation of ATR. The model suggests that ATR localization to a damage site precedes its activation. However, it remains possible that ATR can be activated without localization. Indeed, when TopBP1 is highly overexpressed it activates ATR throughout the nucleus in the absence of a DNA lesion (Fig. ). Furthermore, stable retention of ATR at a damage site is not required for ATR activation, at least, not in response to relatively high doses of UV (3
). Thus, further experiments are required to definitively determine whether localization must precede activation.
ATR, ATM, and other checkpoint signaling pathways are activated by many cancer therapies and regulate the cellular outcomes of these treatments. Disruption of ATR, ATM, and DNA-PK kinases sensitizes cells to radiation and chemotherapy. Since mutations in DNA repair and DNA-damage response pathways are common in cancer cells, these cells are particularly sensitive to disruption of additional pathways. This rationale has driven the development of small molecule inhibitors of DNA damage-responsive kinases for use as chemo- or radio-sensitizing agents (22
). Thus far, specific inhibitors of ATM and DNA-PK kinases have been developed, built around competitive inhibition by binding of ATP analogues (19
). However, specific inhibitors of the ATR kinase have not been found. Unique properties of ATR, such as its requirement for ATRIP, may provide an alternative means of disrupting ATR signaling. Hence, the molecular characterization of ATRIP structure and function as described here may provide a useful starting point for the development of an ATR-targeted therapy.