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ETAA1 - A Newly Discovered Weapon in the Fight Against Replication Stress

By: Carol A. Rouzer, VICB Communications
Published:  October 25, 2016


New research identifies the protein ETAA1 as an activator of ATR, a key player in the DNA damage response to replication stress.


The process of DNA replication is fraught with potential perils, such as difficulty replicating certain sequences, collisions with transcriptional machinery, lack of sufficient nucleotides, and DNA damage. The presence of any of these replication stressors may lead to stalling of the replication fork with recruitment of DNA damage response (DDR) proteins that repair any lesions, correct the problem, and promote resumption of replication (Figure 1). One DDR pathway involves the ATR (ataxia-telangiectasia mutated and RAD3-related) kinase, which is activated through interaction with the single-stranded DNA binding protein RPA (replication protein A) in conjunction with ATRIP (ATR-interacting protein) and TOPBP1 (topoisomerase 2-binding protein 1) (Figure 2). Activation of ATR leads to phosphorylation of CHK1 (checkpoint kinase 1), resulting in cell cycle arrest. This provides time for the cell to resolve the stalled fork before it divides. Now, Vanderbilt Institute of Chemical Biology members David Cortez and Walter Chazin along with their laboratories report the discovery of a new previously unknown pathway for ATR activation [T. E. Bass, et al. (2016) Nat. Cell Biol., published online October 10, DOI:10.1038/ncb3415].



FIGURE 1. There are many causes of replication stress, including difficult sequences, unusual DNA secondary structure, interference with transcriptional machinery, insufficient supply of nucleotides, and DNA lesions. Some pathways used to correct these sources of stress are indicated in bold. Figure reproduced by permission from Macmillan Publishers Ltd, from M. K. Zeman and K.A. Cimprich (2013) Nat. Cell Biol., 16, 2. Copyright 2013.




FIGURE 2. Pathway for ATR activation at a stalled fork. RPA binds to single-stranded DNA exposed at the fork. This leads to recruitment of ATRIP and ATR along with the 9-1-1 complex, which is loaded onto the DNA at the 5′ primer junction by RAD17/RFC2-5. TOPBP1 is then recruited through an interaction with a phosphorylated protein in the 9-1-1 complex. The bound TOPBP1 interacts with and activates ATR which phosphorylates CHK1 and other target proteins. Figure reproduced by permission from Macmillan Publishers Ltd, from K.A. Cimprich and D. Cortez (2008) Nat. Rev. Mol. Cell Biol., 9, 616. Copyright 2008.



Prior work in the Cortez laboratory using the iPOND (isolation of proteins on nascent DNA) technique identified 72 proteins that were enriched at replication forks that had been stalled by exposure of dividing cells to hydroxyurea. Many, but not all of the 72 identified proteins were already known to be involved in the response to replication stress. However, the protein ETAA1 stood out to investigators, as it had not been previously associated with the DDR. The name ETAA1 derives from its prior identification as a Ewing's sarcoma tumor antigen, so the protein was known to be involved in some way with at least one form of cancer. Prior work had also shown that ETAA1 is a target for ATR, as well as ATM, another kinase activated during the DDR. These observations led the researchers to hypothesize that ETAA1 plays a role in the response to replicative stress, and they set out to determine that role.


The investigators found that when they expressed Flag-tagged ETAA1 in U2OS cells, all of the protein was localized in the nucleus. Under normal culture conditions, most of the protein was uniformly distributed throughout the nucleus, but in a small proportion of cells Flag-ETAA1 was concentrated into foci that also contained the DDR protein RPA (Figure 3).


FIGURE 3. Expression of Flag-ETAA1 in U2OS cells. Cells were transfected with an expression vector encoding Flag-ETAA1 and then stained for Flag-ETAA1 (green), RPA (red), or nuclei (blue). ETAA1 was either uniformly dispersed throughout the nucleus (top) or concentrated in foci that also contained RPA (bottom). Figure reproduced by permission from Macmillan Publishers Ltd, from T. E. Bass, et al. (2016) Nat. Cell Biol., published online October 10, DOI:10.1038/ncb3415. Copyright 2016.



Following exposure to a replication stressor, such as camptothecin, cisplatin, or hydroxyurea, accumulation of ETAA1 in foci containing both RPA and γH2AX (a phosphorylated histone that serves as a marker of the DDR) occurred in all cells (Figure 4). These findings support the hypothesis that ETAA1 is involved in the DDR. They also suggest that overexpression of ETAA1 may lead to initiation of a DDR, even in unstressed cells. Notably, accumulation of ETAA1 in foci with RPA and γH2AX did not occur in unstressed cells when the protein was not overexpressed.


FIGURE 4. Expression of Flag-ETAA1 in U2OS cells. Cells were transfected with an expression vector encoding Flag-ETAA1, treated for 3 h with camptothecin (CPT), and then stained for Flag-ETAA1 (green), γH2AX (red, top), RPA (red, bottom), or nuclei (blue). Following CPT, ETAA1 was heavily concentrated in foci that also contained RPA and γH2AX. Figure reproduced by permission from Macmillan Publishers Ltd, from T. E. Bass, et al. (2016) Nat. Cell Biol., published online October 10, DOI:10.1038/ncb3415. Copyright 2016.



Co-immunoprecipitation experiments demonstrated that ETAA1 interacts with RPA. Further studies using fragments of the ETAA1 protein demonstrated that amino acids 571-926 were involved in the RPA interaction. Closer analysis revealed that the sequence of amino acids 900-912 bore close homology to a previously described RPA32C binding motif. Similarly, the sequence of amino acids 600-623 was highly homologous to sequences known to bind to the RPA70N subunit. Deletion of both of these putative binding sites from ETAA1 was required to completely eliminate its association with RPA, suggesting that ETAA1 interacts with both the RPA32C and RPA70N subunits using two distinct binding sites.


The researchers used siRNA to knockdown expression of ETAA1 in cells and CRISPR-Cas/9 to generate an ETAA1 gene knockout cell line (ΔETAA1). In both cases, loss of ETAA1 protein expression resulted in increased  levels of DNA damage markers (RPA and γH2AX), particularly following exposure to camptothecin or hydroxyurea. The cells also exhibited higher levels of DNA double-strand breaks with or without prior exposure to replication stressors. The investigators confirmed increased susceptibility to replication stress in ETAA1-deficient cells by demonstrating slower recovery of DNA synthesis following hydroxyurea exposure, and almost complete failure to resume DNA synthesis following treatment with camptothecin. The researchers also found that they could reverse the effects of ETAA1 knockout by expressing the wild-type protein, but expression of ETAA1 from which the RPA-binding regions had been deleted was only partially effective.


Immunopurification of Flag-ETAA1 from cells revealed co-purification of multiple DDR proteins, including RPA, ATR, and ATRIP, confirming that ETAA1 participates in the formation of one or more large DDR protein complexes. This result also suggested the possibility that ETAA1 is involved in ATR activation. To test this hypothesis, the investigators expressed Flag-tagged fragments of ETAA1, and then used immunoprecipitation to determine which portions of the protein interact with ATR. These experiments demonstrated that an ATR binding site is present in the portion of ETAA1 containing residues 75-250. They noted that in this region of the protein is a sequence that is highly homologous to the ATR-activating domain (AAD) of TOPBP1, and they identified Trp-107 as a key residue within this sequence. Overexpression of residues 75-250 in cells resulted in an accumulation of γH2AX, even in the absence of replication stress, suggesting that this portion of ETAA1 can directly activate the DDR. In contrast, expression of a W107A mutant ETAA1 fragment had no effect on γH2AX levels in the cells. Furthermore, when the researchers used CRISPR-Cas/9 to create a cell line bearing a deletion of exon 2 of the ETAA1 gene, the protein expressed by these cells lacked the putative AAD region and demonstrated similar sensitivity to replication stress as was observed in ΔETAA1 cell lines.


ETAA1 interacts with DDR proteins that are involved in recombination-repair mechanisms, suggesting that ETAA1 may play a role in these processes. Consistently, cells deficient in ETAA1 exhibited increased levels of sister chromatid exchanges (SCEs), a marker of recombination repair failure (Figure 5). This was also observed in cells expressing the exon2 deletion mutant of ETAA1, suggesting that ETAA1-dependent activation of ATR is important in recombination repair.


FIGURE 5. Accumulation of sister chromatid exchanges (SCEs) in U2OS cells following knockdown of BLM, a known recombination repair protein (left), or ETAA1 (right) using siRNA. The SCEs are indicated by the arrows. Figure reproduced by permission from Macmillan Publishers Ltd, from T. E. Bass, et al. (2016) Nat. Cell Biol., published online October 10, DOI:10.1038/ncb3415. Copyright 2016.



As noted above, both ETAA1 and TOPBP1 are capable of activating ATR. Both proteins also share some DDR protein interacting partners. The researchers showed that both ETAA1 and TOPBP1 activate ATR through interaction with ATR's PIKK regulatory domain, and as noted above, the ADDs of ATR and TOPBP1 are similar. These observations suggest that ETAA1 and TOPBP1 might carry out redundant functions. However, the investigators noted that activation of ATR via ETAA1 results in strong phosphorylation of RPA that is not observed when TOPBP1 is the activating protein. Conversely, the CHK1 phosphorylation that occurs when TOPBP1 activates ATR is not observed when activation is effected by ETAA1. Furthermore, while siRNA-mediated knockdown of both ETAA1 and TOPBP1 results in increased SCEs, knockdown of both proteins together leads to even higher SCE occurrence, and siRNA-mediated knockdown of TOPBP1 in ΔETAA1 cells leads to further increases in the cells' sensitivity to camptothecin. These findings suggest that TOPBP1 and ETAA1 activate ATR through independent pathways, serving distinct functions (Figure 6).


FIGURE 6. The results of these studies show that ATR can be activated by ETAA1 (left) or TOPBP1 (right) by two distinct pathways. ETAA1-mediated ATR activation favors phosphorylation of RPA, but other targets remain to be identified. In contrast, when ATR is activated by TOPBP1, phosphorylation of CHK1 is favored. Figure reproduced by permission from Macmillan Publishers Ltd, from T. E. Bass, et al. (2016) Nat. Cell Biol., published online October 10, DOI:10.1038/ncb3415. Copyright 2016.



From these studies we can conclude that ETAA1 is an important player in the response to replication stress. Its primary role appears to be ATR activation following RPA binding. Its ability to directly activate ATR distinguishes it from TOPBP1, which requires the presence of other proteins before it can carry out this function. Indeed, simple overexpression of ETAA1 appears to be enough to activate the DDR, an observation that raises interesting questions concerning its possible role in Ewing's sarcoma. However, before those questions can be addressed, more information is required concerning the targets of ETAA1-activated ATR, a topic for further research.



View Nature Cell Biology article: ETAA1 acts at stalled replication forks to maintain genome integrity






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