Hae Yong Yoo

TopBP1 Activates the ATR-ATRIP Complex

ATR is a key regulator of checkpoint responses to incompletely replicated and damaged DNA, but the mechanisms underlying control of its kinase activity are unknown. TopBP1, the vertebrate homolog of yeast Cut5/Dbp11, has dual roles in initiation of DNA replication and regulation of checkpoint responses. We show that recombinant TopBP1 induces a large increase in the kinase activity of both Xenopus and human ATR. The ATR-activating domain resides in a conserved segment of TopBP1 that is distinct from its numerous BRCT repeats. The isolated ATR-activating domain from TopBP1 induces ectopic activation of ATR-dependent signaling in both Xenopus egg extracts and human cells. Furthermore, Xenopus egg extracts containing a version of TopBP1 with an inactivating point mutation in the ATR-activating domain are defective in checkpoint regulation. These studies establish that activation of ATR by TopBP1 is a crucial step in the initiation of ATR-dependent signaling processes.

Adaptation of a DNA Replication Checkpoint Response Depends upon Inactivation of Claspin by the Polo-like Kinase

The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.

Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses

In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser92 in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser92 in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.

Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM.

ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome.

Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest.

We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks.

The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs.

Aven-Dependent Activation of ATM Following DNA Damage

BACKGROUND In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cells capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.

CtIP interacts with TopBP1 and Nbs1 in the response to double-stranded DNA breaks (DSBs) in Xenopus egg extracts.

In the presence of double-stranded DNA breaks (DSBs), the activation of ATR is achieved by the ability of ATM to phosphorylate TopBP1 on serine 1131, which leads to an enhancement of the interaction between ATR and TopBP1. In Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is additionally required to bridge ATM and TopBP1 together. In this report, we show that CtIP, which is recruited to DSB-containing chromatin, interacts with both TopBP1 and Nbs1 in a damage-dependent manner. An N-terminal region containing the first two BRCT repeats of TopBP1 is essential for the interaction with CtIP. Furthermore, two distinct regions in the N-terminus of CtIP participate in establishing the association between CtIP and TopBP1. The first region includes two adjacent putative ATM/ATR phosphorylation sites on serines 273 and 275. Secondly, binding is diminished when an MRN-binding region spanning residues 25–48 is deleted, indicative of a role for the MRN complex in mediating this interaction. This was further evidenced by a decrease in the interaction between CtIP and TopBP1 in Nbs1-depleted extracts and a reciprocal decrease in the binding of Nbs1 to TopBP1 in the absence of CtIP, suggestive of the formation of a complex containing CtIP, TopBP1, and the MRN complex. When CtIP is immunodepleted from egg extracts, the activation of the response to DSBs is compromised and the levels of ATR, TopBP1, and Nbs1 on damaged chromatin are reduced. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner during the activation of ATR in response to DSBs.