Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.Author SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

Nbn−Mre11 interaction is required for tumor suppression and genomic integrity

We derived a mouse model in which a mutant form of Nbn/Nbs1mid8 (hereafter Nbnmid8) exhibits severely impaired binding to the Mre11−Rad50 core of the Mre11 complex. The Nbnmid8 allele was expressed exclusively in hematopoietic lineages (in Nbn−/mid8vav mice). Unlike Nbnflox/floxvav mice with Nbn deficiency in the bone marrow, Nbn−/mid8vav mice were viable. Nbn−/mid8vav mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn−/mid8 mice developed highly penetrant T cell leukemias. Nbn−/mid8vav leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbnmid8 mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn−/mid8vav malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1. We propose that overexpression of MRE11 compensates for the metastable Mre11−Nbnmid8 interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.

Nbs1 Mediates Assembly and Activity of the Mre11 complex

We derived a mouse model in which a mutant form of Nbs1 (Nbs1mid8) exhibits severely impaired binding to the Mre11-Rad50 core of the Mre11 complex. TheNbs1mid8allele was expressed exclusively in hematopoietic lineages (inNbs1-/mid8vavmice). UnlikeNbs1flox/floxvavmice, which are Nbs1 deficient in the bone marrow,Nbs1-/mid8vavmice were viable.Nbs1-/mid8vavhematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage specific blockage of B cell development. Within six months,Nbs1-/mid8mice developed highly penetrant T cell leukemias.Nbs1-/mid8vavleukemias recapitulated mutational features of human T-ALL, containing mutations inNotch1, Trp53, Bcl6, Bcor, andIkzf1, suggesting thatNbs1mid8mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis ofNbs1-/mid8vavmalignancies showed focal amplification of 9qA2, causing overexpression ofMRE11andCHK1. We propose that overexpression compensates for the meta-stable Mre11-Nbs1mid8interaction, and that selection pressure for overexpression reflects the essential role of Nbs1 in promoting assembly and activity of the Mre11 complex.

Role of the Mre11 Complex in Preserving Genome Integrity

DNA double-strand breaks (DSBs) are hazardous lesions that threaten genome integrity and cell survival. The DNA damage response (DDR) safeguards the genome by sensing DSBs, halting cell cycle progression and promoting repair through either non-homologous end joining (NHEJ) or homologous recombination (HR). The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex is central to the DDR through its structural, enzymatic, and signaling roles. The complex tethers DNA ends, activates the Tel1/ATM kinase, resolves protein-bound or hairpin-capped DNA ends, and maintains telomere homeostasis. In addition to its role at DSBs, MRX/N associates with unperturbed replication forks, as well as stalled replication forks, to ensure complete DNA synthesis and to prevent chromosome rearrangements. Here, we summarize the significant progress made in characterizing the MRX/N complex and its various activities in chromosome metabolism.

Mre11 complex links sister chromatids to promote repair of a collapsed replication fork

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11–Rad50–Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre112–Rad502 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.

Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

TRF2 binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I) and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by hTERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to the I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination (HR) repair, but not HR between sister chromatids or non-homologous endjoining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.Summary StatementTRF2 is recruited to DNA double-strand break damage sites by a two-step mechanism and functions in non-sister chromatid homologous recombination repair

NBS1 is required for macrophage homeostasis and functional activity in mice

Key Points Nbs1 is a component of the MRE11 complex, which is a sensor of DNA double-strand breaks and plays a crucial role in the DNA damage response. In mice with a hypomorphic allele of Nbs1, macrophages exhibit increased senescence and abnormal proliferation and inflammatory responses.