부산대학교 도서관

Cockayne’s Syndrome is a rare hereditary disorder that is characterised by severe neurological abnormalities, cutaneous photosensitivity, severe growth failure and premature aging. The aetiology of this disorder is attributed to mutations and deletions in two proteins, Cockayne’s Syndrome A (CSA) and Cockayne’s Syndrome B (CSB). Both proteins are essential in Transcription-Coupled Nucleotide Excision Repair (TC-NER), a highly specialised pathway that rapidly removes transcription-blocking DNA lesions from the transcribed strand of active genes. Besides a role in TC-NER, CSB has an additional function in general transcription, and both CSA and CSB have been implicated in the repair of oxidative DNA lesions. CSB is known to be ubiquitylated by the CSA ubiquitin ligase complex and at least during TC-NER this process may be coupled to CSB proteosomal degradation. In addition, a recent study from our laboratory identified an ubiquitin-binding domain in CSB, which is essential for its function in TC-NER. The initial aim of the study described in this thesis was to characterise the functional role of protein ubiquitylation in TC-NER through the identification and characterisation of the protein that interacts with CSB’s ubiquitin-binding domain. Realising that this protein might be ubiquitylated CSB itself, an alternate approach was the identification and mutational analysis of ubiquitylation sites in CSB. The latter approach was more successful. The expression and subsequent purification of CSB from human cells facilitated the identification of six ubiquitylation sites. Unexpectedly, mutation of the ubiquitylation site at CSB’s lysine 991 gives rise to genome instability even in the absence of DNA damage. Surprisingly, the CSB K991R cells are proficient in TC-NER, but defective in transcription, and hypersensitive to oxidative damage. To the best of my knowledge, this is the first separation of function mutation described for CSB, and provides new evidence for the possible involvement of de-regulated transcription and persistent oxidative DNA damage in the aetiology of Cockayne’s Syndrome.