RPA-Binding Protein ETAA1 Is an ATR Activator Involved in DNA Replication Stress Response

Abstract

Summary ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. Here, we report the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway via a conserved ATR-activating domain (AAD) located near its N-terminus. Importantly, we demonstrate that both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, our data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

“…As discussed below, this finding directly aligns with the recently published identification of a biochemical function for ETAA1 as an activator of ATR kinase and the replication stress response in human cancer cell lines (18)(19)(20)(21).…”

“…The studies identify RPA-binding domains in the middle and C terminus of ETAA1, and an ATR activation domain in the N terminus encoded by exon 2 and containing a critical W107 residue (corresponding to W109 in mouse). Cancer cell lines lacking ETAA1 had normal rates of cell division but in some but not other cancer lines the loss of ETAA1 resulted in slower and assymetrical progress of replication forks from sites of initiation and slightly increased H2AX Ser139 phosphorylation (18)(19)(20)(21). These biochemical phenotypes of ETAA1-deficient cancer cells were greatly exaggerated by increasing replication stress with hydroxyurea or camptothecin, resulting in overt loss of cell viability.…”

“…Our analysis of mouse mutants with a null allele or an exon 2 deletion allele indicates that in adult animals, ETAA1 is selectively required to suppress the DNA damage response in rapidly proliferating effector T cells during an immune response. These immunologic findings are complemented by four papers published at the end of 2016 identifying ETAA1 as a functional component of the ATR-activating replication stress response complex in human cancer cell lines, with exon 2 encoding the ATR-activating domain of ETAA1 (18)(19)(20)(21). This advance represents a striking demonstration that specific, rate-limiting pathways are needed to insulate proliferating effector T cells against replication stress that might be targeted for suppression of transplant rejection, graft-vs.-host disease, and autoimmune disease.…”

“…The recent studies of ETAA1 in human cancer cell lines reveal that ETAA1 serves as an activator of ATR in parallel with TOPBP1 (18)(19)(20)(21), but likely acts primarily at stalled replication forks with extended ssDNA segments coated by RPA that are too distant to the ssDNA-dsDNA replication junctions for TOPBP1 recruitment. The studies identify RPA-binding domains in the middle and C terminus of ETAA1, and an ATR activation domain in the N terminus encoded by exon 2 and containing a critical W107 residue (corresponding to W109 in mouse).…”

“…Early in the response to virus infection, the rapidly dividing mutant T cells display a strong induction of p53-induced DNA damage response gene sets and increased γH2AX. These results, together with four recent studies demonstrating that ETAA1 associates with RPA at stalled DNA replication forks to stimulate ATR and the replication stress response in nonlymphoid cancer cells in tissue culture (18)(19)(20)(21), reveal a highly specific role for ETAA1 in adult mice regulating replication stress during the formation of effector T cells after infection or immunization.…”