NBS1-CtIP–mediated DNA end resection suppresses cGAS binding to micronuclei

Abstract Cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor—in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor—functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head–associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1’s interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.


Supplementary Figure Legends
A. NBS1 recruitment to micronuclei is independent of nuclear envelope coating. Bar graph shows the frequency of micronuclei containing either nuclear envelope marker (Lamin A/C) alone, NBS1 alone, Lamin A/C and NBS1 or neither in BEAS2B cells treated with 3 µM 6-thio-dG. Data in the bar graph present the mean and STDEV from three independent experiments.

B.
Only a minor fraction of NBS1 co-localizes with RB1 onto micronuclei. Bar graph shows the percentage of micronuclei harboring RB1, NBS1, both or neither in BEAS2B cells treated with 3 µM 6-thio-dG. Data in the bar graph present the mean and STDEV from three independent experiments.
C. Increased proportion of cGAS-positive micronuclei in NBS1 defective cells is not due to micronuclei's defective nuclear envelope coating. Bar graphs show the percentage of micronuclei harboring either Lamin A/C coating (NE) alone, cGAS alone, Lamin A/C and cGAS or neither in BEAS2B cells stably expressing shSCR-and doxycycline-inducible shNBS1 RNAs at 72 hours after 3 µM 6-thio-dG treatment. Bar graphs present the mean and STDEV from three independent experiments. Statistical analysis was performed using Student's t-test.  E. cGAS binds with double-stranded but not to end-resected DNA substrates. 5-10 µM cGAS was incubated with 25 fmol 32 P labeled DNA substrates in the presence of absence of 100 base pairs cold double stranded DNA (competitor). DNA-Protein complex was resolved onto 5% native Poly acrylamide gel electrophoresis and the signal was detected by phosphor imaging. Tables   Table S1: List of primers used for cloning small hairpin RNAs and cGAS binding assay reported in this study.  to these DSBs for chromatin remodeling, end-processing and downstream DDR signaling.

Supplementary
Although ∆MRE11-NBS1 expression in NBS cells did not alter the number of cGAS-positive micronuclei, a previous report indicated a role for MRE11 in sensing cytosolic DNA fragments. 7 However, our results on the lack of co-localization between MRE11 and cGAS within the micronuclei and the cytosolic localization of MRE11 prompted us to investigate MRE11's involvement in the accumulation of cGAS in micronuclei. First, we depleted MRE11 in HT1080 cells by using MRE11-specific shRNA (Fig. S3A). We found that depleting MRE11 did not alter the number of micronuclei that formed in response to 6-thio-dG treatment as compared with control group (Fig. S3B). Furthermore, the accumulation of cGAS in the micronuclei in shMRE11 cells was comparable to that of shSCR cells (Fig. S3C). Additionally, similar to a previous report, 7 the expression of immune signaling genes was reduced in the absence of

MRE11 (Figs. S3E-G).
Second, since MRE11 possesses both exonuclease and endonuclease activities, we used mirin to inhibit MRE11's exonuclease activity to further show that blocking this activity does not augment cGAS accumulation in the micronuclei. As with MRE11 depletion, pre-treating cells with mirin and then with 6-thio-dG did not alter cGAS recruitment to the micronuclei as compared with DMSO-treated cells (Fig. S3D). Additionally, mirin treatment significantly reduced the expression of genes involved in immune pathways in response to 6-thio-dG treatment as compared with 6-thio-dG treated DMSO cells (Fig. S3G). So, MRE11 depletion prevents the induction of an inflammatory response (Figs.S3E-G) even though cGAS recruitment to micronuclei is not altered (Fig. S3D). Thus, cGAS recruited to a limited number of micronuclei in MRE11 depleted cells but does not trigger robust immune response.