Zhao, S.; (2010) Signalling functions of polyubiquitin chains and ubiquitin-binding domains. Doctoral thesis , UCL (University College London).

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Abstract

The ubiquitin signalling system has been shown to regulate many important biological events, ranging from DNA repair to the immune response. Different polyubiquitin chains linked by various linkages have been identified in vivo, and can be recognised by proteins containing ubiquitin-binding domains that act as downstream effectors. However, functions for many of them are not well understood. I have studied the function of K63-linked and linear polyubiquitin chains on a common substrate. The other branch of my study was to investigate the role of ubiquitin binding for a novel ubiquitin-interacting protein, SPC25. K63-linked and linear polyubiquitin chains have a similar topology, but whether they convey a similar signal in vivo remains unclear. I have used the eukaryotic replication clamp PCNA, a natural substrate of K63-linked polyubiquitylation, as a model substrate to directly compare the consequences of modification by different types of polyubiquitin chains. I have shown that K63-polyubiquitylated PCNA is not subject to proteasomal degradation. In contrast, linear, non-cleavable ubiquitin chains do not promote DNA damage tolerance, but function as general degradation signals. I found that a linear tetraubiquitin chain is sufficient to afford proteasomal targeting through the Cdc48-Npl4-Ufd1 complex without further modification. In the second part of my thesis, I describe the identification of SPC25, a subunit of the Ndc80 complex, as a novel ubiquitin-binding protein, using tetra-ubiquitin chains as baits in a genome-wide two-hybrid screen. I have shown that the C-terminal region of SPC25 interacts with ubiquitin in vivo and in vitro. This region does not exhibit significant similarity with any known ubiquitin-binding domains. Further genetic evidence suggests that this ubiquitin-binding domain contributes to the stability of the kinetochore complex.

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