Double-stranded breaks (DSBs) are toxic DNA lesions that if left unrepaired, lead to mutations, chromosomal aberrations, and oncogenesis.The DNA Damage Response (DDR) is a complex network of signaling pathways designed to detect and repair DNA breaks and thus acts as a critical anti-cancer barrier.The MRN complex, consisting of the proteins MRE11, RAD50, and NBS1, is a sensor of DSBs and has roles in facilitating activation of the ataxia-telangiectasia mutated (ATM) kinase, the master regulator of the DDR pathway.ATM phosphorylates a variety of proteins involved in initiating cellular responses important for safeguarding the genome, such as DNA repair, cell cycle checkpoints, and if the damage is too catastrophic to be fixed, apoptosis.A second DNA DSB sensor exists called the KU70/KU80 heterodimer, which binds and activates the kinase DNA-dependent protein kinase, catalytic subunit (DNA-PKcs).To investigate the apparently redundant roles of MRN and KU70/80 in DNA damage kinase signaling, we utilized cells derived from mouse models deficient in either MRN, KU, or both in conjunction with pharmacologic inhibitors for the respective kinases of each DNA damage sensor, ATM and DNA-PKcs.We found that when MRN is deficient, DNA-PKcs effectively substitutes for ATM.Surprisingly, in the absence of both MRN and KU, ATM is still recruited to the chromatin and can facilitate local chromatin responses, including phosphorylation of the histone variant H2AX and recruitment of MDC1.This data implies that MRN is not absolutely required for activation of ATM, as previously thought.Our lab has previously described that the MRE11 C-terminus interacts with cyclin-dependent kinase 2 (CDK2), a kinase important for S-phase cell cycle progression, and that this interaction has roles for homology-directed DSB repair in normally dividing cells.Interestingly, the MRE11 C-terminus is absent in an inherited patient allele causing ataxia-telangiectasia-like disorder 1 (ATLD1).To further understand roles of the MRE11-CDK2 interaction in the DDR, we treated cells with ionizing radiation (IR) to induce DSBs.We found that DNA damage disrupts this interaction in an ATM-dependent manner and causes a reduction in CDK2 catalytic activity.Next, we found that genetic disruption of the interaction in cells lacking either the MRN complex or the MRE11 C-terminus also led to decreased levels of CDK2 activity, mimicking IR-induced disruption of the interaction.Taken together, our data reveal a novel pathway of S-phase checkpoint regulation by the MRE11-CDK2 interaction. To gain greater insight into roles of the MRN complex in cancer, we engineered a mouse model with B lymphocytes lacking MRN or MRN-nuclease activities.Both forms of MRN deficiency led to characteristics of cancer, including oncogenic translocations between C-MYC and the immunoglobulin locus.Surprisingly, these B lymphocytes did not progress to detectable B lineage lymphoma, even in the absence of p53.Moreover, MRE11-deficiencies prevented tumorigenesis in a mouse model strongly predisposed to spontaneous B-cell lymphomas.Our findings indicate that MRN is not a classic tumor suppressor and instead imply that certain functions of the MRN complex, such as MRE11 nuclease activity, are required for oncogenesis.These data also suggest that MRE11 nuclease activity can be targeted for cancer therapy development.
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Regulation of ATM and CDK2 Kinases by the MRN Complex in DNA Damage Signaling and Cell Cycle Checkpoint Control
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