Mistakes in the repair of damaged DNA can lead to conditions such as disorders of the nervous system, increased genetic instability and cancer. Thousands of single-stranded DNA breaks occur in cells on a daily basis; if mistakes are made in their repair, disease is a likely outcome. One of the proteins that functions in the repair of single-stranded DNA breaks is Aprataxin (APTX), a unique member of the histidine triad (HIT) superfamily. APTX is a neurodegenerative disease-related protein whose dysfunction causes ataxia with oculomotor apraxia 1 (AOA1), a relatively rare condition that is characterized by progressive problems with movement, especially coordination, sufferers often losing the ability to walk. It is now known that APTX has a direct role in catalyzing the nucleophilic release of adenylate groups from 5’-AMP-termini at DNA nicks or breaks during the repair of abortive DNA ligation. This discovery of the DNA deadenylase activity of APTX provides a molecular rationale for AOA1. To date, the mechanism underlying the reversal of 5’-adenylated DNA by aprataxins has remained puzzling as no structures of these enzymes bound to DNA have been described.　　 

In a recent paper published in Nature Structural & Molecular Biology (Crystal structures of aprataxin ortholog Hnt3 reveal the mechanism for reversal of 5′-adenylated DNA, Nature Structural & Molecular Biology 18, 1297–1299, 2011), Professors Da-Cheng Wang and Tao Jiang and colleagues at the Institute of Biophysics, Chinese Academy of Sciences report the crystal structures of the yeast Aprataxin ortholog apo Hnt3, and Hnt3-DNA and Hnt3-DNA-AMP complexes at 1.8, 1.9 and 2.1 angstrom resolution, thus shedding light on how this APTX protein recognizes and processes 5’ adenylated DNA in a structure-specific manner. The structures show that Hnt3 uses a rigid, preformed surface to interact with dsDNA which provides a distinct molecular platform for DNA recognition and processing and consists of three essential parts: a unique DNA-binding cleft which binds the 5’ end of the short DNA, a ZF domain which contacts the long DNA strand in a sequence-independent manner, and a conserved HIT active site which specifically interacts with the 5’ AMP site. The structures reveal the molecular mechanism of DNA deadenylation that resolves abortive ligation. The dsDNA adopts a 5’-flap conformation that facilitates 5’-AMP access to the active site, where AMP cleavage occurs by a previously proposed two-step catalytic mechanism in which the enzyme first breaks the phosphodiester bond of the substrate by transferring AMP to a histidine residue of the HIT motif, and then activates a water molecule to hydrolyze the phosphohistidine bond to release AMP and regenerate the enzyme. In addition, these structures shed light on the substrate specificity of Hnt3 as a DNA nick sensor, explaining why aprataxins prefer to bind 5’-adenylated nicks and double-stranded breaks. The results reported support the idea that the HIT-ZF domain is sufficient for efficient DNA processing, and that the FHA domain of APTX may coordinate interactions with other proteins during DNA repair. The structure-based conclusions from this work are generally consistent with results from a series of biochemical studies, suggesting that the structures of the complexes reported in this paper reflect the physiological assembly mode for DNA deadenylation.

Professors Wang, Jiang and colleagues also identified some key amino acid residues involved in Hnt3 function through mutational and interaction studies, and showed that different Aprataxin mutations affect DNA repair in different ways. This report thus, in addition to providing a structural framework for understanding the mechanism of DNA deadenylation, provides important insights into the molecular basis of ataxia with oculomotor apraxia 1.

Figure. Crystal structure of Hnt3 in complex with double-stranded DNA and AMP.