Mutations in the ubiquitin binding UBZ motif of DNA polymerase η do not impair its function in translesion synthesis during replication

Treatment of Saccharomyces cerevisiae cells with DNA-damaging agents elicits lysine 164-linked PCNA monoubiquitination by Rad6-Rad18. Recently, a number of ubiquitin (Ub) binding domains (UBDs) have been identified in translesion synthesis (TLS) DNA polymerases and it has been proposed that the UBD in a TLS polymerase affects its binding to Ub on PCNA and that this binding mode is indispensable for a TLS polymerase to access PCNA at the site of a stalled replication fork. To evaluate the contribution of the binding of UBDs to the Ub moiety on PCNA in TLS, we have examined the effects of mutations in the C₂H₂ zinc binding motif and in the conserved D570 residue that lies in the α-helix portion of the UBZ domain of yeast Polη. We find that mutations in the C₂H₂ motif have no perceptible effect on UV sensitivity or UV mutagenesis, whereas a mutation of the D570 residue adversely affects Polη function. The stimulation of DNA synthesis by Polη with PCNA or Ub-PCNA was not affected by mutations in the C ₂H₂ motif or the D570 residue. These observations lead us to suggest that the binding of Ub on PCNA via its UBZ domain is not a necessary requirement for the ability of polymerase η to function in TLS during replication.