Final Report Summary - MOBA-CS (The molecular basis of Cockayne Syndrome)
The evolutionarily conserved CUL4 E3 ligases, in concert with their DDB1 adaptor, regulate a diverse set of cellular processes including development, transcription, replication and DNA repair. Within the CRL4DDB2 ligase, DDB2 recruits the ligase to damaged DNA. The remainder of the ligase receptors serve in pathways unrelated to DNA damage recognition. CRL4 specificity is conferred upon by a set of more than 14 WD40 containing substrate receptors, also referred to as DCAFs (DDB1 CUL4 Associated Factors). Using the CRL4DDB2 ligase as a model system, we studied targeting and regulation of this bi-functional nano-machine, investigating common architectural and functional traits amongst CRL4 ligases. We solved the CRL4DDB2 ligase structure examining its architecture and function through a combination of structural biology, cell biology and biochemistry. On the biochemical level, we were able to reconstitute, for the first time, the entire CRL4 activation process from DNA damage binding to ligase activation comprising in excess of 18 polypeptides. We showed, by structural means that the CUL4 ligase moiety within the CRL4DDB2 complex is mobile, and freely rotates 60° to 120° around damage. This flexible architecture prevents CUL4 from sensing whether DDB2 has engaged with damaged DNA duplexes. Instead, we found that the observed DNA damage dependent activation of CRL4DDB2 is mediated by the COP9 signalosome (CSN). We also solved the structure of DDB1 bound to the Cockayne syndrome A (CSA) protein. CSA is a WD40 containing DCAF receptor that is functionally unrelated to DDB2. This structure showed that the DDB2 substrate receptor can be exchanged with CSA, and indeed other WD40 substrate adaptors, retaining the overall architecture of the CRL4 complex. Different WD40 containing substrate receptors can thus be interchanged in a “plug-and-play” fashion, giving rise to ligases with similar architecture but different specificity. This work let to the investigation of Cop9 signalosome and general mechanism of CRL ligase regulation and provided a first molecular basis for mutations in Cockayne syndrome.