Novel ubiquitin binding sites of S. cerevisiae polymerase η identified by a genetically encoded photoactive probe (927.4)

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DNA translesion synthesis is carried out by specialized DNA polymerases such as Polη. Polη contains a ubiquitin‐binding zinc finger (UBZ) domain required for its effective recruitment to the stalled replication fork in response to DNA damages. Our previous study reported a novel ubiquitin‐binding mode of S. cerevisiae Polη in which the C‐terminal portion of Polη binds ubiquitin with 30‐fold higher affinity than the UBZ domain alone. However, the mechanism of such higher binding affinity is not clear. In this study, a genetically encoded photoactive probe, p‐benzoyl‐L‐phenylalanine (pBpa), was incorporated at various positions on ubiquitin to map the interaction sites between Polη and ubiquitin. Data suggests that besides the Ile44 hydrophobic patch which was known to bind UBZ domain of Polη, several other residues at the ubiquitin C‐terminus and a surface opposite from the hydrophobic patch on ubiquitin also bind to Polη. These new binding sites increase the binding affinity between ubiquitin and Polη. Our finding is important for understanding the role of ubiquitin in the recruitment of Polη during DNA translesion synthesis.Grant Funding Source: Supported by U.S National Science Foundation Grant MCB‐0953764 to Z. Zhuang