Rad6/Rad18 Competes with DNA Polymerases η and δ for PCNA Encircling DNA

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 the following: if Rad6/Rad18 utilizes the universal binding sites (or nearby sites), then PCNA monoubiquitination may be suppressed by pols involved in TLS. Results from quantitative studies reveal that (1) a Y-family pol (pol η) and a B-family pol (pol δ) critical to TLS each inhibit the transfer of ubiquitin from Rad6/Rad18 to PCNA and that (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.