Ubiquitin-binding motif of human DNA polymerase η is required for correct localization

Acharya et al. (1) recently examined the role of different motifs in human DNA polymerase (pol)η. Their data suggested that mutations in the ubiquitin-binding (UBZ) motif of polη had no effect on its localization into replication foci. However, the first 4 authors of this Letter have independently found, in many experiments, that mutations D652A, C638A, and H654A in the UBZ motif all greatly reduced the accumulation of polη in replication foci (refs. 2 and 3 and our unpublished data). Acharya et al. (1) used cells cotransfected with GFP-PCNA and FLAGpolη, whereas we analyzed the localization of eGFPpolη with endogenous PCNA. Fig. 1 demonstrates unequivocally that this is the cause of the discrepancy. Transfection of eGFPpolη–D652A alone failed to form foci, as reported previously (2). In contrast, when cotransfected with mRFP-PCNA, eGFPpolη–D652A did form foci, as shown by Acharya et al. (1). We conclude that the UBZ motif of polη is required for correct localization of polη when cellular concentrations of PCNA are unperturbed. If, however, the concentration of PCNA is artificially increased, the UBZ of polη becomes dispensable. Neither our data nor those of Acharya et al. (1) provide evidence that the ubiquitinated species to which the UBZ motif binds in the nuclear foci is ubiquitinated PCNA. In fact, we have recently shown that ubiquitination of PCNA appears not to be required for polη accumulation into foci, but it does increase the residence time within foci, suggesting the existence of multiple signals that are required for controlling the dynamics of UBZ-containing proteins in the nucleus (4). Wrnip1 provides a further example in which mutations in the UBZ motif abolished localization into foci (5).

Fig. 1.

MRC5 cells were transfected with either wild-type or D652A mutant eGFPpolη without (Uppers) or with (Lowers) mRFP-PCNA. Twenty-four hours later, the cells were UV-irradiated (30 Jm⁻²) and incubated for 6 h. Cells were fixed in paraformaldehyde and examined directly for epifluorescence or incubated with methanol in those samples which were analyzed for endogenous PCNA by immunofluorescence.