Evidence for multiple functions of Aprataxin in DNA damage repair

Bellstedt, Peter

This dissertation addresses the structural and functional characterization of human Aprataxin, a DNA damage repair protein with a unique activity not found for any other protein involved in DNA repair. Single strand breaks (SSBs) threaten genomic stability, as more than 10.000 of such SSBs arise per cell and day. During the repair of SSBs, so-called "dirty" DNA breaks may arise, which cannot be ligated resulting in the formation of an abortive ligation intermediate: 5'-adenylated DNA. The 5'-adenylate cannot be processed by Ligase 3 itself but requires Aprataxin to be removed from the DNA. Aprataxin belongs to the superfamily of histidine triad (HIT) proteins, but its domain organization including a catalytically active HIT motif and a zinc finger (ZnF) is unique. This work is focused on the HIT-ZnF due to its functional significance. A structural characterization of HIT-ZnF was hampered by its high precipitation propensity, precluding structure determination in solution via standard NMR methodology. By utilizing the specific individual unlabeling of all 20 amino acids, 78% of the observable backbone resonances could be assigned. Based on the assigned chemical shifts a structural CS-ROSETTA-based model of the HIT-ZnF domain of human Aprataxin is presented. The resonance assignment also provides a basis for a detailed characterization of the interaction between HIT-ZnF, its substrate(s) and small chemical compounds by NMR spectroscopy in the future. Surprisingly, functional analysis revealed that HIT-ZnF har- bors additional nucleolytic activities on ribonucleotide-containing substrates different from the canonical adenylated DNA. Although the functional significance of these in vitro observations has yet to be evaluated in vivo, this work provides a solid basis for a comprehensive future assessment of Aprataxin's activities and a role beyond resolving an abortive ligation intermediate during single-strand break repair.

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Bellstedt, Peter: Evidence for multiple functions of Aprataxin in DNA damage repair. 2014.

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