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Studies on protein:protein interactions in mammalian DNA base-excision repair

Dulic, Anna; (2003) Studies on protein:protein interactions in mammalian DNA base-excision repair. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Repair of damaged DNA is required to maintain the integrity of genetic information. DNA repair processes employ numerous proteins whose efficient function depends on interactions with each other. The major pathway that corrects endogenous DNA damage is base-excision repair (BER), which is mediated by sequential pair-wise protein interactions. In this study, I have used various techniques to investigate interactions mediating the DNA synthesis/ligation steps of BER, to better understand the way in which DNA repair processes are co-ordinated. The interaction of DNA ligase III with its protein partner, XRCC1, in BER was the first example of a functional interaction between BRCT modules, and the 3D structure of this XRCC1 BRCT domain has been solved. The 'BRCT' domain, first noted in the C-terminus of the product of the BRCA1 breast cancer suppressor gene (BRCA1 Carboxy Terminal), has been identified in~50 proteins involved in DNA repair/recombination/cell cycle control. I have used DNA ligase III-XRCC1 as a model system to study BRCT domains. Amino acid changes in the BRCT domain of XRCC1 have been introduced by site-specific in vitro mutagenesis of the cDNA sequence, and the interaction with DNA ligase III has been tested by co-affinity precipitation assays. The consequences of mutations on protein folding were also assessed. Reciprocal mutations in DNA ligase III were made to test the validity of using the XRCC1 BRCT domain structure to model other BRCT modules. I have also investigated a possible role of a major 3' exonuclease activity, DNase III, as a candidate DNA-editing activity for DNA polymerase β (which lacks an intrinsic 3' exonuclease function) during BER. Far Western, co-affinity, co-immunoprecipitation and yeast two- hybrid analyses were employed to identify protein: protein interactions that might confirm this role. Novel interactions indicate that DNase III might have a specialised role in the immune system.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Studies on protein:protein interactions in mammalian DNA base-excision repair
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; DNA damage repair
URI: https://discovery.ucl.ac.uk/id/eprint/10097636
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