Proteomics reveals a new DNA repair factor involved in DNA damage signaling

ABSTRACT

Stalling of the DNA replication machinery activates the ATR checkpoint kinase, which coordinates the cellular responses to replication stress. New studies identify a novel ATR activator, ETAA1, which is indispensable for the maintenance of genome integrity. Dysregulation of ETAA1 may contribute to the development of pancreatic cancer.

DNA replication occurs with extraordinary speed and precision. However, in certain situations the processivity of the replication machinery is impaired, for example when the replication machinery runs into DNA lesions or when nucleotides are limiting. Because faithful segregation of the newly replicated DNA into daughter cells can only occur when every segment of the genome has been fully replicated, any stalling of the replication machinery immediately triggers an alarm signal to pause cell cycle progression so that problems at the affected forks can be resolved.¹

How do cells recognize these stalled forks? At every replication fork the discontinuous mode of replication exposes about 70–250 nucleotides of single-stranded DNA (ssDNA) on the lagging strand template² that are immediately covered by replication protein A (RPA), a heterotrimeric complex composed of RPA1–3. Upon fork stalling, the persistent RPA-ssDNA filament acts as a recruiting platform for a large number of proteins involved in DNA metabolism and DNA damage signaling. However, RPA-bound ssDNA also serves as an intermediate in several DNA repair pathways, including long-patch base excision repair, mismatch repair, and pathways required for the repair of DNA double-strand breaks. How cells distinguish between these various RPA-ssDNA filaments and regulate recruitment of the cognate repair factors remains largely unknown.

To gain insight into this process, we and others have recently established unbiased proteomic methods that allow comprehensive surveillance of the recruitment of proteins to RPA-ssDNA formed under various conditions. This led to the parallel identification and characterization of a hitherto poorly studied protein called Ewing's Tumor Associated Antigen 1 (ETAA1) in studies reported back-to-back in a recent issue of Nature Cell Biology.^{3, 4} Intriguingly, ETAA1 interacts with RPA via 2 RPA binding motifs called RBM1 and RBM2, which bind to conserved pockets on RPA1 and RPA2, respectively (Fig. 1A). The binding pocket on RPA1 additionally mediates interactions with a number of DNA checkpoint proteins including the ATR Interacting Protein (ATRIP), RAD9, and MRE11,⁵ whereas the RPA2 binding pocket accommodates peptide sequences from a diverse set of RPA-interacting proteins, including XPA, SMARCAL1, and UNG2. At first glance, this might suggest that ETAA1 could function as a competitive inhibitor to dampen the checkpoint response by sterically blocking access of other proteins to the RPA-coated ssDNA. However, this is not the case; in fact, ETAA1 activates the ATR/ATRIP protein kinase through direct binding. Thus, ETAA1 activity provides a short-cut alternative to the well-established ATR activation mechanism, which involves the sensing of a free 5´-OH group at nearby ssDNA/dsDNA junctions and therefore requires additional checkpoint factors such as the Topoisomerase II Binding Protein 1 (TOPBP1), CLASPIN, and the RAD9/RAD1/HUS1 (9–1–1) complex (Fig. 1B).^{6, 7} Strikingly, both ETAA1 and TOPBP1 contain a conserved ATR activation domain (AAD) that is sufficient to stimulate purified ATR in vitro.

Figure 1.

ETAA1 accumulates on RPA-coated ssDNA and activates the ATR kinase. (A) Two RPA binding motifs (RBM1 and RBM2, blue) interact with conserved binding pockets on RPA1 and RPA2, respectively. The same binding pockets also accommodate similar peptides of other DNA checkpoint and repair factors. Conserved amino acid residues of human RPA interactors are shown above. The structural models show the amino- and carboxyterminal domains of RPA1 and RPA2 with a bound inhibitor or peptide (PDB file 5e7n and 1dpu, respectively). (B) Model for 2 parallel ATR activation pathways. Left: Direct activation by ETAA1, Right: Canonical pathway involving TOPBP1 and the RAD9, RAD1, HUS1 (9–1–1) complex. ATR, ataxia telangiectasia and Rad3-related; ETAA, Ewing's tumor associated antigen 1; RPA, replication protein A.

Although ETAA1 deletion affects phosphorylation of only a subset of ATR substrates, it causes strong cellular phenotypes including hypersensitivity to hydroxyurea, campthothecin, and mitomycin C and increased frequencies of various mitotic errors. Furthermore, the synthetic lethality observed upon TOPBP1 depletion in ETAA1-deficient cells suggests that ETAA1 and TOPBP1 act in distinct pathways. Whether certain routes to checkpoint activation require only one or both of these activators remains to be determined. So far, the strongest ETAA1 accumulation was observed upon inhibition of the replicative polymerase by aphidicolin or upon depletion of the nucleotide pool with hydroxyurea, which both lead to the formation of extensive stretches of ssDNA. Remarkably, however, ETAA1 also accumulates at replication forks stalled at DNA interstrand crosslinks⁸ or even at resected DNA double-strand breaks in the absence of any active replication forks.⁴ More work will be required to understand the distribution of labor between ETAA1 and TOPBP1 in the activation of ATR under these various conditions.

It will be interesting to further analyze how the dual interaction mode of ETAA1 with the RPA complex determines the cellular response to replication stress. On the one hand, ETAA1 may link 2 neighboring RPA molecules, thereby increasing the stability of the ssDNA-RPA filament. On the other hand, dual interaction may allow more flexibility in regulating access of proteins to the RPA-coated ssDNA. For example, if ATRIP displaced ETAA1 from the RPA1 binding pocket, ETAA1 could nevertheless retain its interaction with the ssDNA-RPA filament via the RPA2 binding pocket. Determining the stoichiometry of the various factors on ssDNA under different conditions as well as their post-translational modifications will be required to fully understand the regulation of protein recruitment to the RPA-coated ssDNA filament. Given the intricate binding mode, we anticipate that ETAA1 might play a key role in coordinating such recruitment and in channeling ssDNA intermediates into the appropriate downstream processes.

Although ETAA1 was discovered under replication stress conditions, it may also be important during unperturbed DNA replication. ETAA1 readily accumulates on undamaged chromatin in a strictly replication-dependent manner (our unpublished observation). Consistent with this notion, deletion of ETAA1 not only dramatically reduces replication fork speed, but also leads to a higher frequency of asymmetric replication tracts. These dramatic changes in the replication dynamics suggest that in the absence of ETAA1 replication forks frequently collapse and give rise to some of the spontaneously occurring mitotic aberrations.

Our findings reveal a pivotal role for ETAA1 at DNA replication forks and RPA-coated ssDNA tracts formed during the repair of DNA double-strand breaks. ETAA1 contributes to the sensing of stalled replication forks via activation of the ATR/ATRIP kinase. In addition, ETAA1 likely regulates the recruitment of other DNA repair factors to RPA-coated ssDNA. Our mechanistic studies provide a possible explanation for why mutations at the ETAA1 gene locus are linked with an increased risk of pancreatic cancer.⁹ Understanding the precise function of ETAA1 may also be important for rationalized cancer treatment, as cell-permeable small-molecule inhibitors have been identified that bind to the RPA1 binding pocket and thereby block protein interactions and checkpoint activation.¹⁰ Such inhibitors might prove useful as chemosensitizers in combination with already approved chemotherapeutics.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Niels Mailand, Zuzana Storchova, Neysan Donelly, Mario Avarello, and Narendra Chunduri for comments on the manuscript and Christian Biertümpfel for help with the figure.

Funding

This work was supported by the Center for Integrated Protein Science, Munich