de Moura Miguez Araújo, Sofia Jorge; (2000) Interactions and function of nucleotide excision repair complexes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nucleotide excision repair (NER) is the main pathway by which mammalian cells remove damage to their DNA caused by UV light and some other mutagens. The initial stages of the NER reaction involve recognition of DNA lesions and cleavage of the damaged DNA strand on either side of the modified base(s) resulting in the release of 24-32-mer oligonucleotides containing the damage (dual incision). The duplex DNA molecule is then restored by semi-conservative DNA synthesis and ligation (repair synthesis). In order to more fully define which factors are required for dual incision formation I have reconstituted the NER incision reaction using exclusively recombinant proteins on a defined DNA substrate containing a single 1,3-d(GTG) cisplatin adduct. The minimal set of factors necessary for this reaction is RPA, XPA, TEIIH complex (without the CAK kinase subunits), XPC-hHR23B complex, XPG, and ERCCl-XPE complex. TEIIH containing CAK, probably the most physiologically relevant form, can also function in NER. To further define the whole NER reaction, I reconstituted DNA repair synthesis using the recombinant and the highly purified human proteins RPA, RFC, PCNA, DNA ligase I and DNA polymerases δ or ϵ. Additionally, I purified a factor from HeLa cells, IF7, that significantly stimulates NER incision reactions and found that it is neither necessary for the incision stages of NER nor for repair synthesis. Xeroderma pigmentosum group D (XP-D) cells, defective in XPD helicase subunit of TEIIH, generally have a relatively high level of UV- induced unscheduled DNA synthesis even though they are deficient in NER. To investigate whether this is due to altered 5' or 3' incision activity we studied both XP-D and TTD cells using in vitro NER assays. The majority of XP-D cell extracts tested (bearing different mutations in the XPD gene) were defective in both 3' and 5' incisions, though some TTD cell lines made dual incisions weakly. Purified TFIIH containing a defined mutation in the XPD subunit was active in transcription but did not make 3' or 5' incisions around a cisplatin lesion. To investigate the existence of a human repairosome and repair subcomplexes, immunoprecipitation and functional assays were used. In human cells, a functional association consisting of all the above mentioned factors except RPA can be detected at low ionic strength. The strongest interaction found is between TFIIH and XPC.

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