Cryo-EM structure of nucleotide-bound Tel1ATM unravels the molecular basis of inhibition and structural rationale for disease-associated mutations

Yeast Tel1 and its highly conserved human ortholog ataxia-telangiectasia mutated (ATM) are large protein kinases central to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia (A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using cryoelectron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our structure reveals molecular details of key residues surrounding the nucleotide binding site and provides a structural and molecular basis for its intrinsically low basal activity. We show that the catalytic residues are in a productive conformation for catalysis, but the phosphatidylinositol 3-kinase-related kinase (PIKK) regulatory domain insert restricts peptide substrate access and the N-lobe is in an open conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also provides a structural rationale for many mutations found in A-T and cancer

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 DNA 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 severely impaired 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

Regulation of the Yeast Cell Cycle Checkpoint Kinase Tel1 at Double-Strand DNA Breaks and at Telomeres

The work discussed in this dissertation focuses on two distinct areas that are critical for genome maintenance. Both areas are focused on the regulation of the Saccharomyces cerevisiae checkpoint kinase Tel1 by the MRX complex. Tel1 kinase initiates cell cycle checkpoint in response to double-strand DNA breaks. Tel1 also plays a major role in telomere maintenance. Tel1’s function both in checkpoint signaling and telomere regulation is dependent on the Mre11-Rad50-Xrs2 complex. In Chapter II, I describe a robust biochemical approach aimed at reconstituting the initial stages of double-strand DNA break response using purified proteins in order to address how the MRX complex and DNA orchestrate to activate Tel1 kinase. Our results demonstrate that double-stranded DNA and MRX activate Tel1 synergistically. This work revealed a DNA length dependent stimulation of Tel1, with long, nucleosome-free duplex DNA being the preferred effector for full Tel1 activation. Our work also highlights there is no requirement for double-stranded DNA ends for Tel1 activation by MRX in vitro. Our data show Rad50 is the most critical subunit as no stimulation of Tel1 is seen with the Mre11-Xrs2 pair and DNA, which is in agreement with reported genetic observations. This stimulatory effect of Rad50 is absolutely dependent on its ATP binding activity. This work provides a comprehensive model into how individual subunits of MRX collaborate with DNA to activate Tel1 kinase. The second portion of this thesis discusses work employing the biochemical assay described in Chapter II to investigate the regulation of Tel1 kinase and MRX at telomeres. Similar to several other checkpoint and DNA repair factors, Tel1 and MRX are involved in telomere regulation. Unique architectural organization and remodeling of telomeric DNA by telomere binding proteins prevents telomeres from inappropriate recognition and repair as intrachromosomal DNA breaks. In budding yeast, Rif2 is one of the main proteins involved in checkpoint response suppression at long telomeres. MRX has been shown to recruit Tel1 specifically to short telomeres, resulting in the recruitment of telomerase and the elongation of short telomeres. Conversely, based on genetic observations Rif2 has been suggested to attenuate elongation of long telomeres through the MRX-Tel1 pathway. Our studies show Rif2 directly inhibits MRX-dependent activation of Tel1 kinase. The inhibitory role of Rif2 is mediated through its conserved N-terminal domain. Our data demonstrate that Rif2 exerts its inhibition by modulating the ATPase activity of Rad50. Investigation of an allosteric Rad50 ATPase mutant that maps outside of the conserved ATP binding domain suggest Rif2 discharges the ATP-bound form of Rad50, which is a state conducive for Tel1 activation. Taken together these data point to a novel role of Rif2 in regulating Tel1 kinase through the Rad50 subunit of M