BACKGROUND AND PURPOSEThe D1CT-7 mouse is one of the best known animal models of Tourette syndrome (TS), featuring spontaneous tic-like behaviours sensitive to standard TS therapies; these characteristics ensure a high face and predictive validity of this model, yet its construct validity remains elusive. To address this issue, we studied the responses of D1CT-7 mice to two critical components of TS pathophysiology: the exacerbation of tic-like behaviours in response to stress and the presence of sensorimotor gating deficits, which are thought to reflect the perceptual alterations causing the tics. EXPERIMENTAL APPROACHD1CT-7 and wild-type (WT) littermates were subjected to a 20 min session of spatial confinement (SC) within an inescapable, 10 cm wide cylindrical enclosure. Changes in plasma corticosterone levels, tic-like behaviours and other spontaneous responses were measured. SC-exposed mice were also tested for the prepulse inhibition (PPI) of the startle response (a sensorimotor gating index) and other TS-related behaviours, including open-field locomotion, novel object exploration and social interaction and compared with non-confined counterparts. KEY RESULTSSC produced a marked increase in corticosterone concentrations in both D1CT-7 and WT mice. In D1CT-7, but not WT mice, SC exacerbated tic-like and digging behaviours, and triggered PPI deficits and aggressive responses. Conversely, SC did not modify locomotor activity or novel object exploration in D1CT-7 mice. Both tic-like behaviours and PPI impairments in SC-exposed D1CT-7 mice were inhibited by standard TS therapies and D1 dopamine receptor antagonism. CONCLUSIONS AND IMPLICATIONSThese findings collectively support the translational and construct validity of D1CT-7 mice with respect to TS. AbbreviationsNC, non-confined; PPI, prepulse inhibition of the acoustic startle reflex; SC, spatial confinement; TS, Tourette syndrome
Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2'-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide synthesis. These studies yield an improved understanding of the mutational origins of 2'-modified substrate recognition as well as identify SFM19 as the best candidate for further engineering, whether via rational design or directed evolution.
DNA polymerases enable key DNA biotechnologies by performing enzymatic amplification of DNA with high efficiency and fidelity, enabling DNA's use in polymerase chain reaction (PCR), sequencing techniques and many different applications in therapeutics and diagnostics. While these astounding properties of DNA polymerases enable the use of these technologies, they also restrict them because they do not recognize modified substrates. Three previous studies have evolved DNA pol I from Thermus aquaticus (Taq) to recognize 2’ modifications of DNA; while these have resulted in an increase in modified substrate recognition, these enzymes are not active enough for practical use. Here, we describe efforts to identify the key mutations that impart unnatural activity on these previously identified enzymes. In particular, we have focused on mutations at I614 and E615 since they occur in all three previously identified mutants. Data show that much of the activity that is present in the previously evolved enzymes is largely a result of the E615G mutation. Mutations at I614 have been found to have no effect when present alone, but have also been found to have varying effects on enzymatic activity when in conjunction with the E615 mutation. Current work is also being done to further investigate the synergistic interactions of mutations of I614 and E615G. These efforts will elucidate key features of the evolution of unnatural substrate recognition by mutant Taq enzymes and will aid in future polymerase engineering efforts.
In addition to encoding all known life, DNA is also an invaluable biotechnological tool. Chemical modifications to DNA, such as 2’ modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2’ modified DNA nucleotides. While these enzymes recognize these modified nucleotides, they are not capable of synthesizing DNA containing more than six to eight modified nucleotides. Here, we describe comparative biochemical studies that seek to better understand these previously evolved enzymes through quantitative characterization. We describe the identification of specific amino acids and combinations of amino acids that contribute most to unnatural activity. Notably, we have also found that these enzymes are capable of using substrates beyond the originally selected substrates, introducing the possibility of new evolved enzyme‐modified substrate pairs. Collectively, through quantitative characterization of mutations, substrate specificity, extension efficiency and structure, we hope to elucidate key features of previously evolved DNA polymerases. These studies should inform future DNA polymerase engineering efforts, whether via rational design or directed evolution.
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