title: A novel ubiquitin binding mode in the S. cerevisiae translesion synthesis DNA polymerase η.
abstract: The ubiquitin binding zinc finger (UBZ) domain in the C-terminal portion of Polη has been found to interact with ubiquitin. However, the affinity between the Polη UBZ and ubiquitin was shown to be low with a previously reported K(d) of 73-81 μM. This low-affinity binding between Polη 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 Polη UBZ domain and probed the Polη UBZ-ubiquitin interaction using a surface plasmon resonance (SPR) technique. Our quantitative binding data between the wild-type or mutant Polη UBZ and ubiquitin revealed an interesting divergence between the Polη UBZ from S. cerevisiae and humans. Moreover, we found that the C-terminal portion of yeast Polη (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 Polη and the isolated UBZ domain. This observation raised the interesting possibility that the Polη 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.
authors.affiliation: Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware, Newark, DE 19716, USA.
authors.forename: Yongxing
authors.initials: Y
authors.lastname: Ai
authors.forename: Jialiang
authors.initials: J
authors.lastname: Wang
authors.forename: Robert E
authors.initials: RE
authors.lastname: Johnson
authors.forename: Lajos
authors.initials: L
authors.lastname: Haracska
authors.forename: Louise
authors.initials: L
authors.lastname: Prakash
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D057075:Enzyme Assays; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D015032:Zinc; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases