Structure-function studies on 5′ nucleases

The 5′ nucleases are members of the Flap endonucleases (FENs) family of structure-specific DNA-processing metalloenzymes. They fulfil essential functions DNA replication and repair. Genome integrity relies on their inherent 5′ to 3′ exonuclease and 5′ flap endonuclease activities. A variety of bifurcated nucleic acids can be processed in vitro, hence these enzymes have become important component of many molecular biology techniques.

Catalytic parameters for the T7 gene product 6 exonuclease (T7 gp6), a FEN family member, were determined for various substrates using a fluorescent real-time assay. Binding affinity was determined by electrophoretic mobility shift assay. In attempts to modulate relative and absolute levels of endonuclease and exonuclease activity, the impact of buffer composition and site-directed mutagenesis of residues in and around the active site was explored. The effect on catalytic activity and DNA binding affinity of mutations in the T7 gp6 active-site was studied. Three mutations retained DNA binding affinity comparable to the wild-type enzyme. Two of which were catalytically inert (Asp160Lys, Asp162Lys) whilst the third (Asp202Lys) exhibited severely reduced nuclease activity. Mutation in the potassium ion-binding helix-3-turn-helix motif (Ile200Arg) increases the specificity constant (kcat/KM) compared to wild-type for all substrates tested by at least 2-fold. Additionally, T7 gp6 Ile200Arg exhibited enhanced binding affinity for single-flap substrate but not a nicked (exonuclease) substrate, compared to wild type, suggesting the helix-3-turn helix motif contributes to differential modulation of endonuclease and exonuclease activity.

Attempts to determine the structure of T7 gp6 failed. However, a high-resolution structure (1.44 Å) of T5FEN active-site mutant Asp155Lys and two structures of Taq polymerase FEN domain with DNA were solved (1.82 –2.18 Å). The latter represent novel structural insights into how this important enzyme interacts with DNA.