Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex.

The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA.

Chemical methods for protein site-specific ubiquitination.

Ubiquitination is an important protein post-translational modification regulating many cellular processes in eukaryotes. Ubiquitination is catalyzed by a three-enzyme cascade resulting in the conjugation of the C-terminal carboxylate of ubiquitin (Ub) to the ε-amino group of a lysine residue in the acceptor protein an isopeptide bond. enzymatic ubiquitination utilizing Ub ligases has been successfully employed to generate Ub dimers and polymers.

Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes.

Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellular processes. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating enzymes (DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinated proteins as substrates, more direct evidence is needed to support this notion.