Activation of the ATR kinase by the RPA-binding protein ETAA1

ABSTRACT: The ATR kinase is a master regulator of cellular responses to DNA damage and replication stress that is activated by a complex mechanism involving its binding partner ATRIP, RPA-coated ssDNA, the 9-1-1 complex and TopBP1. Here, we discovered a new ATR activation mechanism in human cells, mediated by the uncharacterized protein ETAA1 (Ewing’s tumor-associated antigen 1). From a comprehensive proteomic survey of protein recruitment to DNA double-strand break-modified chromatin, we identified ETAA1 as a factor that accumulates at DNA damage sites via an RPA-binding motif, and which has an important role in promoting cell survival and preventing replication fork breakage after genotoxic insults. Mechanistically, we show that ETAA1 harbors a conserved domain that potently and directly stimulates ATR kinase activity independent of TopBP1 and 9-1-1, and which is essential for the function of ETAA1 in the DNA damage response. Consistently, ablation of ETAA1 shows profound synthetic lethality with TopBP1 knockdown, resulting from massive replication fork collapse and abrogation of ATR-dependent signaling. Finally, we show that ETAA1 levels in cancer cell lines inversely correlate with their dependency on TopBP1 for ATR activation, and that overexpression of ETAA1 partially restores ATR-dependent signaling after loss of TopBP1. Together, these findings establish a new mechanism of ATR activation in human cells that operates in parallel with, but independent of, the canonical TopBP1-mediated pathway.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Xenopus Laevis (african Clawed Frog)

TISSUE(S): Egg, Cell Culture, Early Embryonic Cell

SUBMITTER: Mario Oroshi  

LAB HEAD: Matthias Mann

PROVIDER: PXD003529 | Pride | 2016-09-30

REPOSITORIES: Pride