Translesion DNA synthesis (TLS) bypasses DNA lesions encountered during S-phase and is critical for cell survival after exposure to DNA-damaging agents. In humans, Rad6/Rad18 attaches single ubiquitin moieties (i.e. monoubiquitination) to proliferating cell nuclear antigen (PCNA) sliding clamps encircling primer/template (P/T) junctions that are stalled at DNA lesions. TLS occurs via PCNA monoubiquitination-independent and -dependent pathways and both contribute to cell survival. The interaction of Rad6/Rad18 with PCNA is paramount to PCNA monoubiquitination and remains poorly defined. In particular, the location of the Rad6/Rad18 binding site on PCNA is unknown. Many PCNA-binding proteins, particularly DNA polymerases (pols), converge on PCNA encircling stalled P/T junctions in human cells and all interact in a similar manner with the universal binding sites on PCNA. We reasoned that if Rad6/Rad18 utilizes the universal binding sites (or nearby sites), then PCNA monoubiquitination may be suppressed by pols involved in TLS. In this study published in Biochemistry, we characterized the effects of a Y-family (pol η) and a B-family (pol δ) human DNA polymerase on monoubiquitination of PCNA encircling DNA by utilizing a novel fluorescence assay developed in our lab. Results from extensive, quantitative studies reveal that; 1) pols η and δ, both of which are critical to TLS, inhibit transfer of ubiquitin from Rad6/Rad18 to PCNA and; 2) the observed inhibitions are dependent on the interaction of these pols with PCNA encircling DNA. These studies suggest that Rad6/Rad18 utilizes the universal PCNA-binding sites or nearby sites and, hence, competes for PCNA encircling DNA with pols η and δ and possibly other PCNA-binding proteins involved in TLS. These findings provide valuable insight into the nature of the interaction between Rad6/Rad18 and PCNA and have important implications for the division of human TLS pathways. Check out the full article here: https://pubs.acs.org/doi/10.1021/acs.biochem.9b00938