Here we use structural studies to determine how two different deubiquitinating enzyme (DUB) complexes deal with K63-linked ubiquitin chains in DNA repair.
The BRCA1-A complex is involved in pathway choice for dsDNA break repair. Here we study how this DUB complex acts on K63-linked ubiquitin chains. We designed a metallo-DUB probe for K63 ubiquitin chains and used it to obtain cryo-EM structure of BRCA1-A with K63-chains. These cryo-EM structures show how BRCA1-A recognises K63 polyubiquitin chains and suggests the origin of the BRCA1 A preference for longer chains.
DNA damage tolerance (DDT) allows bypass of DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, as mono-ubiquitination of PCNA initiates recruitment of TLS polymerases and serves as substrate for K63-linked poly-ubiquitination that is necessary for template switching mediated bypass. We studied how USP1/UAF1 cleaves ubiquitin chains on PCNA, using kinetic analysis of differentially labelled ubiquitin chains. Mechanistic insights suggest how this could influence the choice of DDT pathway.