Structural and Functional Studies of hAPTX

Abstract

<p>DNA integrity is continuously compromised by cellular metabolic activity and environmental factors resulting in many lesions per cell per day (Lindahl et al.,2009). If left unrepaired or if repaired improperly, these lesions pose an obstacle to cellular transcription and replication. In due course, DNA damage may lead to mutations and genomic instability that will eventually threaten the viability of the cell and organism and can lead to tumour formation and neurological disorders (Rass et al., 2006). Aprataxin (Aptx) is a conserved factor in DNA repair required for the repair of damaged DNA following abortive DNA ligation (Date et al., 2001; Moreira et al 2001). Aptx initiates repair by processing adenylated DNA ligation events via its unique AMP lysine hydrolase activity (Rass et al 2006). Inability of Aptx to carry out these functions has been shown to be the causative agent in the neurological diseases, ataxia with oculomotor apraxia (Takahashi et al 2007, Yoon et al 2008, and Ferrarini et al., 2007). Nevertheless, the exact mechanism of Aptx has not been established and the full extent of its function(s) (i.e. binding to DNA or other proteins) is not fully understood. In order to achieve a comprehensive understanding of the mechanism governing Aptx, further functional and structural analysis is required. Here, it was found that Aptx has deadenylation activity that is further supported by the crystal structure of ATPX-DNA-AMP-Zn²⁺. Importantly, work reported in this thesis describes for the first time methods for expression and purification of large quantities of high quality human Aptx from bacterial cells. This protein is shown to possess robust deadenylation activity, suitable for further <em>in vitro</em> screening of small molecule inhibitors.</p>