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

The Y-family DNA polymerase eta (Pal eta) 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 Poll) catalytic core and PCNA have been solved, a high-resolution structure of the complex of Pol eta/PCNA or Pol eta/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Poly) C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photoactivatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (papa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Pol eta, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Pol eta catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Pol eta in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.

Automated summary

The study used photoactivatable PCNA and Ub-PCNA probes to identify direct interactions of the Pol η catalytic core with both sides of the PCNA ring in the polymerase complex. This provides important insights for investigating PCNA-containing complexes involved in DNA replication, repair, and damage tolerance.