Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability

Abstract

The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9–based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle– and replication stress–dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.

Mus81-Eme1–dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity

“…Replication stress often results in chromosome mis-segregation ( 6 , 16 , 23 , 33 ). We then asked whether excess origin firing and fork slowdown result in chromosome mis-segregation in Chk1-deficient cells [phenotypes in S and M phases were determined 48 and 72 hours after transfection with small interfering RNA (siRNA), respectively; fig.…”

“…Our study suggests that nucleotide starvation in Chk1-depleted cells impedes the accurate handling of UR-DNA, boosting genomic instability, manifested as anaphase aberrations, micronuclei, UFBs, and 53BP1-NBs. Distortion of dNTP pools, mitotic onset before completion of DNA duplication, and abnormal anaphases have been associated with cell death in cells lacking DDR proteins, either ATR, WEE1, or ETAA1 ( 20 , 23 , 55 ). Intriguingly, however, we found no such causal connection between genomic instability and cell death in Chk1-depleted cells ( Fig.…”

“…DNA under-replication leads to nascent DNA synthesis in early mitosis, i.e., mitotic DNA synthesis (MiDAS). The inactivation of various DDR effectors induces UR-DNA and MiDAS (20)(21)(22)(23)(24). However, it remains unknown whether Chk1 loss induces UR-DNA and MiDAS and whether mitotic events prevent or promote CIN in Chk1-deficient cells.…”

ETAA1 ensures proper chromosome segregation: A matter of S phase or mitosis?

“…Achuthankutty et al (11) conducted a CRISPR-Cas9 screen to identify genes whose ablation is synthetically lethal with ETAA1 loss. Gene ontology analysis indicated a notable enrichment of terms associated with DNA replication among the genes identified.…”

“…1). Unlike Achuthankutty et al (11), whose screening approach focused on identifying synthetic lethal interactions with ETAA1, Bass and Cortez (9) used quantitative phosphoproteomics to identify factors functioning downstream of ETAA1. This approach unveiled a prominent role of ETAA1 during mitosis, as many kinetochore- and spindle-localized proteins were phosphorylated in an ETAA1-dependent but TopBP1-independent manner.…”