Ewing Tumor-associated Antigen 1 Interacts with Replication Protein A to Promote Restart of Stalled Replication Forks

Abstract

The replication protein A (RPA) complex binds singlestranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.The faithful replication of DNA is essential for the maintenance of genomic stability and the prevention of cancerpromoting mutations. Replication forks can be stalled by numerous obstacles on the DNA template, including unrepaired DNA damage, DNA-bound proteins, and secondary structures (1). Stalled replication forks are able to restart once the obstacles are removed or become broken (collapse) into DNA double strand breaks, which pose the most serious threat to genome integrity when fork protection fails (2, 3). However, how stalled replication forks are protected is not well understood.The replication protein A (RPA) 3 complex, which consists of RPA1 (RPA70), RPA2 (RPA32), and RPA3 (RPA14), plays crucial roles in a variety of DNA metabolic pathways, including DNA replication, recombination, repair, and DNA damage checkpoint (4 -6). When replication forks stalled, singlestranded DNA (ssDNA) is generated and extended by minichromosome maintenance protein complex helicases (7,8). The ssDNA is bound by RPA, which protects ssDNA from cleavage by nucleases and recruits repair proteins to initiate DNA damage responses. The RPA-ssDNA complex recruits and activates ATR/ATRIP thereby eliciting checkpoint signaling (9). In addition, RPA-ssDNA complex also recruits factors necessary for the stabilization and resumption of stalled replication forks, such as RAD51 (10, 11) and SMARCAL1 (12-15). Recently, several studies have also revealed a physical and functional interaction between RPA and the ubiquitin E3 ligases RFWD3 (16 -18) and PRP19 (19), which ubiquitinate RPA and facilitate replication fork restart. Here, we identified a new RPA interaction protein, ETAA1, whose gene variation has been associated with susceptibility to pancreatic cancer (20). ETAA1 is recruited to stalled replication forks in response to replication stress, and the disruption of ETAA1 leads to fork colla...

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.…”