PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

Abstract

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18•RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.

The RPA complex orchestrates K63-linked deubiquitination via ZUP1

“…We find that the ZUP1 a-2/3 region, the potential S1' site in ZUP1, is important for ZUP1-RPA complex formation, though other interaction sites are likely based on our XL-MS data, suggesting a more complex mode of binding compared to other RPA-interacting partners such as SMARCAL1 and p53 (Extended Data Figure 4D). This is reminiscent of the E3 ligase RAD18, which has been shown to bind to multiple subunits within RPA and its activity is regulated by the interaction with RPA 31,45,46 . Importantly, we show that RPA functions to stimulate the DUB activity of ZUP1 against K63-linked polyUb chains.…”

High resolution studies of DNA lesion bypass by human DNA polymerase δ holoenzymes

“…Human RPA, Cy5-PCNA, RFC, and pol δ (exonuclease-deficient and wild-type) were obtained as previously described (15,16). The concentration of active RPA was determined via a FRET-based activity assay as described previously (17).…”

DNA replication: Mechanisms and therapeutic interventions for diseases

“…196 PCNA is mono-ubiquitinated at K164 by RAD6-RAD18 E2-E3 complex in response to replication fork stalling. 197 Mono-ubiquitinated PCNA increases affinity with polη, thus further promoting efficient TLS. 198 Hence, dysregulation of replication and TLS progression cause severe genomic instability and tumorigenesis.…”

“…PCNA is mono‐ubiquitinated at K164 by RAD6–RAD18 E2–E3 complex in response to replication fork stalling 197 . Mono‐ubiquitinated PCNA increases affinity with polη, thus further promoting efficient TLS 198 .…”