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The XPC-hHR23b complex and nucleotide excision repair of DNA

Batty, Dawn Patricia; (1999) The XPC-hHR23b complex and nucleotide excision repair of DNA. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

In human cells, nucleotide excision repair (NER) removes DNA damage caused by UV light and acts on a variety of other helix-distorting adducts. Xeroderma pigmentosum group C (XP-C) cells are deficient in NER and are complemented by a 125 kDa polypeptide (XPC) which is found complexed with a yeast Rad23 homologue, hHR23B. XPC (pl25) and XPC-hHR23B complex were expressed in insect cells, hHR23B was expressed in E. coli and all were purified. Both XPC and XPC-hHR23B were active in complementing XPC-deficient cell extracts. In a reconstituted system using other purified NER factors, XPC alone possessed only weak repair activity and hHR23B gave a 10-fold stimulation. An electrophoretic mobility-shift assay was set up in order to obtain the first quantitative measurements of the preference of XPC complex for damaged DNA. XPC-hHR23B had a 400-fold preference for binding UV damaged DNA over non damaged, a discrimination similar to that determined here in parallel for the UV damaged DNA binding factor, UV-DDB. Binding of XPC-hHR23B to UV damaged DNA showed a marked temperature-dependence. Association was detectable within seconds even at 0° C. Once bound, the damaged DNA- XPC-hHR23B complexes were stable and dissociation was slow. At least 50 % of the complexes were detectable 6 h after challenge with excess UV-damaged DNA at 30° C. XPC-hHR23B had a high affinity for (6-4) photoproducts as damage binding was little affected by enzymatic photoreactivation of the UV- irradiated probe. Mobility-shift competition assays showed that XPC-hHR23B had some affinity for DNA treated with cisplatin and alkylating agents. Both non and UV damaged single-stranded M13 were extremely efficient competitors of XPC-hHR23B binding. Synthetic single-stranded homopolymer DNA was a relatively poor competitor. This suggests that XPC-hHR23B interacts with structures formed within single-stranded or damaged DNA of mixed sequence. XPC-hHR23B may act as a major damage recognition factor in human NER.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The XPC-hHR23b complex and nucleotide excision repair of DNA
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest
Keywords: Pure sciences; DNA repair
URI: https://discovery.ucl.ac.uk/id/eprint/10107791
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