Abstract
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase functions as a central node in the DNA damage response signaling network. The mechanisms by which ATR activity is amplified and/or maintained are not understood. Here we demonstrate that BRIT1/microcephalin (MCPH1), a human disease-related protein, is dispensable for the initiation but essential for the amplification of ATR signaling. BRIT1 interacts with and recruits topoisomerase-binding protein 1 (TopBP1), a key activator of ATR signaling, to the sites of DNA damage. Notably, replication stress-induced ataxia telangiectasia-mutated or ATR-dependent BRIT1 phosphorylation at Ser-322 facilitates efficient TopBP1 recruitment. These results reveal a mechanism that ensures the continuation of ATR-initiated DNA damage signaling. Our study uncovers a previously unknown regulatory axis of ATR signaling in maintaining genomic integrity, which may provide mechanistic insights into the perturbation of ATR signaling in human diseases such as neurodevelopmental defects and cancer.
Highlights
ATR signaling depends on topoisomerase-binding protein 1 (TopBP1) recruitment to replication stress sites
We showed that FLAG-BRIT1 could pull down endogenous TopBP1 (Fig. 1A) and that there is a physical interaction between endogenous TopBP1 and BRIT1 (Fig. 1B)
We showed that DNA did not mediate this interaction because BRIT1 was coimmunoprecipitated with TopBP1 even after DNase I treatment (Fig. 1C)
Summary
ATR signaling depends on TopBP1 recruitment to replication stress sites. Results: Interaction between BRIT1 and TopBP1 is required for sustained TopBP1 recruitment to sites of replication stress, which maintains ATR substrate activation. Replication stress-induced ataxia telangiectasia-mutated or ATR-dependent BRIT1 phosphorylation at Ser-322 facilitates efficient TopBP1 recruitment. These results reveal a mechanism that ensures the continuation of ATR-initiated DNA damage signaling. Autophosphorylation of ATR on RPA-coated, single-stranded DNA enables interaction between ATR and TopBP1 to stimulate ATR kinase activity and facilitate recognition of the substrates of ATR [12, 13] It remains to be answered how ATR signaling is maintained/amplified when ATR is activated. The presence of phosphorylated H2AX (␥-H2AX) provides docking sites to recruit DNA damage-responsive sensors, such as NBS1 and MDC1, through their phosphoprotein-interacting BRCA1 C terminus (BRCT) domains, and recruitment of these sensor proteins further activates or maintains ATM kinase activity and amplifies ATM signaling [17,18,19]. In the study reported here, we found that BRIT1 functionally interacts with the ATR activator TopBP1 and is required for the continuation of ATR signaling
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