Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

Abstract

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…In rd-NHEJ, an initial resection step involves MRE11 exonuclease, EXD2, Plk3-phosphorylated CtIP/BRCA1, EXO1, and Artemis endonuclease before repair through C-NHEJ occurs. MRE11 endonuclease activity may be dispensable in rd-NHEJ (92), whereas in MMEJ, initial resection involves both endo- and exonuclease activity of the MRN complex plus other enzymes, such as CtIP, PARP1, FEN1, and DNA ligases I and III (93, 94). The minimum homology for ligation is ∼5 bp (94).…”

“…MRN subunit biochemical activities depend upon their three-dimensional folds, but MRN biological functions are governed by its complexes and conformationally regulated interaction networks. Thus, HR and MMEJ are activated in irradiated human cells owing to phosphorylation-dependent formation of either NBS1 or XRCC1 repair complexes with MRE11–RAD50 (93). In fact, the use of the same MRN subunits in multiple response networks provides a physical means to coordinate sensing, signaling, chromatin remodeling, and repair processing.…”

NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation

“…Strikingly, CtIP phosphorylation was not strictly required in the absence of NBS1 (Figs 5C-G and 8B). Nevertheless, CtIP likely activates MRE11 in MMEJ, which is not restricted to S-G2 and requires only minimal resection Averbeck et al, 2014;Sharma et al, 2015;Dutta et al, 2017). In yeast, Sae2 phosphorylation regulates its physical interaction with RAD50 , in addition to Xrs2 (Liang et al, 2015).…”

Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair

“…To gain a deeper understanding of the pathways involved in zygotic IHR, we performed a series of injections in Cas9-injected Chd2 R1684H/+ zygotes with multiple DSB repair- and homologous recombination (HR)-associated proteins: USP1/WDR48 (promotes HR via Fanconi-Anemia (FA) pathway 25 ), BCCIP (promotes BRCA2-mediated HR 26-28 , participates in FA pathway 29 ), XRCC1 (promotes MMEJ and co-localizes with RAD51 30,31 ), XRCC4 (promotes NHEJ and regulator of V(D)J recombination 32,33 ), and DMC1 (meiotic HR regulator 34 ). XRCC1, XRCC4, and DMC1 were not capable of inducing IHR at rates above baseline ( Fig.…”