The ubiquitin variant UbKEKS modifies PCNA to enhance DNA polymerase processivity during replication

Proliferating Cell Nuclear Antigen (PCNA) is a pivotal regulator of DNA replication and repair, orchestrating protein recruitment through its binding domains and post-translational modifications (PTMs). Lysine 164 (K164) of PCNA serves as a critical molecular switch modulated by ubiquitin (Ub) and ubiquitin-like proteins. Here, we identify the ubiquitin variant Ub ^KEKS as a new PCNA regulator, demonstrating its covalent modification of K164 and quantifying the Ub ^KEKS -to-Ub modification ratio. Loss of Ub ^KEKS results in delayed S-phase progression without altering PCNA levels or nuclear localization. However, DNA fiber combing assays reveal a significant reduction in DNA polymerase processivity in Ub ^KEKS -deficient cells, indicating its role in replication efficiency. At a whole-cell scale, the mapping by mass spectrometry of diGly remnants after trypsin digestion, demonstrate that modifications by Ub ^KEKS or Ub do not compensate one another, due to key differences regarding amino acids surrounding modified lysines. Ultimately, our findings establish Ub ^KEKS as a distinct key modulator of PCNA function, expanding the repertoire of PTMs that influence DNA replication dynamics. These insights pave the way for further exploration of Ub ^KEKS as a regulator of genome stability and cell cycle regulation.