ABSTRACT
The DNA damage response (DDR) is orchestrated by three apical signalling kinases: ATM, ATR and DNA-PKcs. Despite their central roles, structural and biochemical understanding has remained limited, mainly due to their large size. Recent advances in cryo-electron microscopy allowed for the structural analysis of these kinases, revealing their overall architecture and providing high resolution structures of their active sites. Combined with novel biochemical insights it is now possible to dissect the elements that are important for activation. In this review we discuss the recent structures of these kinases, the possible mechanisms to regulate their activity, substrate recognition and emerging insights in the elements required for kinase activation.
Subject(s)
DNA Damage , Signal Transduction , Ataxia Telangiectasia Mutated Proteins/metabolism , Cryoelectron Microscopy , DNAABSTRACT
Tel1 (ATM in humans) is a large kinase that resides in the cell in an autoinhibited dimeric state and upon activation orchestrates the cellular response to DNA damage. We report the structure of an endogenous Tel1 dimer from Chaetomium thermophilum. Major parts are at 2.8 Å resolution, including the kinase active site with ATPγS bound, and two different N-terminal solenoid conformations are at 3.4 Å and 3.6 Å, providing a side-chain model for 90% of the Tel1 polypeptide. We show that the N-terminal solenoid has DNA binding activity, but that its movements are not coupled to kinase activation. Although ATPγS and catalytic residues are poised for catalysis, the kinase resides in an autoinhibited state. The PIKK regulatory domain acts as a pseudo-substrate, blocking direct access to the site of catalysis. The structure allows mapping of human cancer mutations and defines mechanisms of autoinhibition at near-atomic resolution.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/metabolism , Chaetomium/enzymology , Adenosine Triphosphate/metabolism , Catalytic Domain , Chaetomium/chemistry , DNA/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Homeostasis , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protein MultimerizationABSTRACT
Sister chromatid cohesion is mediated by cohesin, whose Smc1, Smc3, and kleisin (Scc1) subunits form a ring structure that entraps sister DNAs. The ring is opened either by separase, which cleaves Scc1 during anaphase, or by a releasing activity involving Wapl, Scc3, and Pds5, which bind to Scc1 and open its interface with Smc3. We present crystal structures of Pds5 from the yeast L. thermotolerans in the presence and absence of the conserved Scc1 region that interacts with Pds5. Scc1 binds along the spine of the Pds5 HEAT repeat fold and is wedged between the spine and C-terminal hook of Pds5. We have isolated mutants that confirm the observed binding mode of Scc1 and verified their effect on cohesin by immunoprecipitation and calibrated ChIP-seq. The Pds5 structure also reveals architectural similarities to Scc3, the other large HEAT repeat protein of cohesin and, most likely, Scc2.
