Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18

Thach T, et al., 54(12):2136-48, Biochemistry, 2015

Described herein is the mechanism by which Rad18 recognizes polyubiquitin at a molecular level and how it functions in the DNA damage response (DDR) pathway. A Pall ForteBio BLItz instrument was used to perform Bio-Layer Interferometry (BLI) studies. For linkage-specific diubiquitin binding to Rad18 experiments, amine reactive sensor tips were immobilized with linear, K48-linked, or K63-linked Ub2. Ni-NTA biosensors immobilized with His-Ub, His-Ub2, His-Ub3, or His-Ub4 were used to investigate length-dependent binding of linear polyubiquitin chains to Rad18. The KD values were determined by the steady state analysis of Req values. The results demonstrate continuous ubiquitin binding domains (comprising UBZ and ELRM of Rad18) outcompete RAP80 in polyubiquitin recognition, suggesting a potential regulation of the DDR pathway.

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