Jeffrey Buis

Mre11 Nuclease Activity has Essential Roles in DNA Repair and Genomic Stability Distinct from ATM Activation

The Mre11/Rad50/NBS1 (MRN) complex maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution but play unknown roles in mammals. To define the functions of Mre11, we engineered targeted mouse alleles that either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from the absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology-directed double-strand-break repair and a contributing role in activating the ATR kinase. However, the nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair, distinct from MRN control of ATM signaling.

Multiple functions of MRN in end-joining pathways during isotype class switching

The Mre11–Rad50–NBS1 (MRN) complex has many roles in response to DNA double-strand breaks, but its functions in repair by nonhomologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in class switch recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end, we have engineered mice that lack the entire MRN complex in B lymphocytes or that possess an intact complex that harbors mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a strong defect in CSR, affecting both the classic and the alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the classical and alternative NHEJ pathways.

Mre11 regulates CtIP-dependent double-strand break repair by interaction with CDK2

Homologous recombination facilitates accurate repair of DNA double-strand breaks (DSBs) during the S and G2 phases of the cell cycle by using intact sister chromatids as sequence templates. Homologous recombination capacity is maximized in S and G2 by cyclin-dependent kinase (CDK) phosphorylation of CtIP, which subsequently interacts with BRCA1 and the Mre11–Rad50–NBS1 (MRN) complex. Here we show that, in human and mouse, Mre11 controls these events through a direct interaction with CDK2 that is required for CtIP phosphorylation and BRCA1 interaction in normally dividing cells. CDK2 binds the C terminus of Mre11, which is absent in an inherited allele causing ataxia telangiectasia–like disorder. This newly uncovered role for Mre11 does not require ATM activation or nuclease activities. Therefore, functions of MRN are not restricted to DNA damage responses but include regulating homologous recombination capacity during the normal mammalian cell cycle.

Recruitment and activation of the ATM kinase in the absence of DNA-damage sensors.

Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11−Rad50−NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70−Ku80). Using mouse cells containing targeted mutant alleles of Mre11 (Mre11a) and/or Ku70 (Xrcc6), together with pharmacologic kinase inhibition, we demonstrate that ATM can be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. In the absence of both MRN and Ku, ATM is recruited to chromatin, where it phosphorylates H2AX and triggers the G2-M cell-cycle checkpoint, but the DNA-repair functions of MRN are not restored. These results suggest that, in contrast to straightforward recruitment and activation by MRN, a complex interplay between sensors has a substantial role in ATM control.

Disease-associated MRE11 mutants impact ATM/ATR DNA damage signaling by distinct mechanisms

DNA double-strand breaks (DSBs) can lead to instability of the genome if not repaired correctly. The MRE11/RAD50/NBS1 (MRN) complex binds DSBs and initiates damage-induced signaling cascades via activation of the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia- and rad3-related (ATR) kinases. Mutations throughout MRE11 cause ataxia-telangiectasia-like disorder (ATLD) featuring cerebellar degeneration, and cancer-predisposition in certain kindreds. Here, we have examined the impact on DNA damage signaling of several disease-associated MRE11A alleles to gain greater understanding of the mechanisms underlying the diverse disease sequelae of ATLD. To this end, we have designed a system whereby endogenous wild-type Mre11a is conditionally deleted and disease-associated MRE11 mutants are stably expressed at physiologic levels. We find that mutations in the highly conserved N-terminal domain impact ATM signaling by perturbing both MRE11 interaction with NBS1 and MRE11 homodimerization. In contrast, an inherited allele in the MRE11 C-terminus maintains MRN interactions and ATM/ATR kinase activation. These findings reveal that ATLD patients have reduced ATM activation resulting from at least two distinct mechanisms: (i) N-terminal mutations destabilize MRN interactions, and (ii) mutation of the extreme C-terminus maintains interactions but leads to low levels of the complex. The N-terminal mutations were found in ATLD patients with childhood cancer; thus, our studies suggest a clinically relevant dichotomy in MRE11A alleles. More broadly, these studies underscore the importance of understanding specific effects of hypomorphic disease-associated mutations to achieve accurate prognosis and appropriate long-term medical surveillance.

MRE11 promotes tumorigenesis by facilitating resistance to oncogene-induced replication stress

Hypomorphic mutations in the genes encoding the MRE11/RAD50/NBS1 (MRN) DNA repair complex lead to cancer-prone syndromes. MRN binds DNA double-strand breaks, where it functions in repair and triggers cell-cycle checkpoints via activation of the ataxia-telangiectasia mutated kinase. To gain understanding of MRN in cancer, we engineered mice with B lymphocytes lacking MRN, or harboring MRN in which MRE11 lacks nuclease activities. Both forms of MRN deficiency led to hallmarks of cancer, including oncogenic translocations involving c-Myc and the immunoglobulin locus. These preneoplastic 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 cannot be considered a standard tumor suppressor and instead imply that nuclease activities of MRE11 are required for oncogenesis. Inhibition of MRE11 nuclease activity increased DNA damage and selectively induced apoptosis in cells overexpressing oncogenes, suggesting MRE11 serves an important role in countering oncogene-induced replication stress. Thus, MRE11 may offer a target for cancer therapeutic development. More broadly, our work supports the idea that subtle enhancements of endogenous genome instability can exceed the tolerance of cancer cells and be exploited for therapeutic ends. Cancer Res; 77(19); 5327-38. ©2017 AACR.

Abstract 2489: Mre11-CDK2 interaction during the DNA double-strand break response

The MRN (Mre11/Rad50/NBS1) complex is a group of highly conserved proteins central to the detection and repair of DNA double-strand breaks (DSBs). Inherited deficiencies in DNA DSB signaling result in a spectrum of disorders featuring cancer predisposition, neurodegeneration, and immunodeficiency. MRN directly binds DNA DSB ends and facilitates activation of ataxia-telangiectasia mutated (ATM) kinase, which initiates cellular responses including DNA repair, cell cycle checkpoints, and in some circumstances, apoptosis. Cellular responses to DNA DSBs require rapid communication between DNA repair complexes and the cell cycle machinery, but much about this relationship remains unclear. Our lab recently discovered an interaction between Mre11 and Cyclin-dependent kinase 2 (CDK2), a key component of the cell cycle machinery (Buis et al., NSMB, 2012). CDK2, when bound to its regulatory partner Cyclin A, promotes cell cycle progression through S phase. We demonstrated that the Mre11-CDK2/Cyclin A interaction is important in regulating the capacity for DNA repair by homologous recombination in normally dividing cells. Current work is focused on understanding the role of this interaction during DNA damage responses. We hypothesize that Mre11 interaction with CDK2 is a component of a molecular switch between the normal cell cycle and the DNA damage response. To test our hypothesis, we take advantage of murine alleles of Mre11 previously generated in the lab. We performed immunoprecipitation studies to investigate the protein-protein interaction in the presence of DNA damage. We also utilized kinase inhibitors to investigate the role of apical kinases in regulating the interaction. Finally, we are currently optimizing in vitro kinase assays to further understand a relationship between Mre11 and CDK2 enzymatic activity.Unpublished findings reveal that the Mre11-CDK2/Cyclin A interaction is altered upon DNA damage, a process which appears to be important for cell cycle checkpoint regulation. In conclusion, we demonstrate that the Mre11-CDK2 interaction plays important roles both in normally dividing cells and in the DNA damage response. Taken together, our data suggests that Mre11 may act to bridge the gap between DNA repair complexes and the cell cycle machinery. Citation Format: Mary J. Morgan, Todd A. Festerling, Jeffrey Buis, David O. Ferguson. Mre11-CDK2 interaction during the DNA double-strand break response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2017-2489