A novel ubiquitin binding mode in the S. cerevisiae translesion synthesis DNA polymerase η

The ubiquitin binding zinc finger (UBZ) domain in the C-terminal portion of Pol eta has been found to interact with ubiquitin. However, the affinity between the Pol eta UBZ and ubiquitin was shown to be low with a previously reported K(d) of 73-81 mu M. This low-affinity binding between Pol eta UBZ and ubiquitin has been difficult to reconcile with its presumed role in translesion synthesis as suggested by genetic and cell biology studies. In this work, we constructed a minimal S. cerevisiae Pol eta UBZ domain and probed the Pol eta UBZ-ubiquitin interaction using a surface plasmon resonance (SPR) technique. Our quantitative binding data between the wild-type or mutant Pol eta UBZ and ubiquitin revealed an interesting divergence between the Pol eta UBZ from S. cerevisiae and humans. Moreover, we found that the C-terminal portion of yeast Pol eta (amino acid 515-632) binds ubiquitin with a much higher affinity than the minimal UBZ domain. Further, distinct ubiquitin-binding kinetics were observed for the C-terminal portion of Pol eta and the isolated UBZ domain. This observation raised the interesting possibility that the Pol eta C-terminal portion binds ubiquitin in a novel mode that affords higher affinity. Our findings have broader implication in understanding the generally weak interaction between the known ubiquitin-binding domains and ubiquitin.