4 research outputs found
Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI
Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 310 helix and containing β-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal ‘arms’ that appear to ‘compete’ for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate
Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence
Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters
Increasing cleavage specificity and activity of restriction endonuclease KpnI
Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent
metal ions for DNA cleavage but not for binding. The active site of KpnI can
accommodate metal ions of different atomic radii for DNA cleavage. Although
Mg2+ ion higher than 500 μM mediates promiscuous activity, Ca2+ suppresses the
promiscuity and induces high cleavage fidelity. Here, we report that a
conservative mutation of the metal-coordinating residue D148 to Glu results in
the elimination of the Ca2+-mediated cleavage but imparting high cleavage
fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved
through better discrimination of the target site at the binding and cleavage
steps. Biochemical experiments and molecular dynamics simulations suggest that
the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry
of the Ca2+-bound HNH active site. Although the D148E mutant reduces the
specific activity of the enzyme, we identified a suppressor mutation that
increases the turnover rate to restore the specific activity of the high
fidelity mutant to the wild-type level. Our results show that active site
plasticity in coordinating different metal ions is related to KpnI promiscuous
activity, and tinkering the metal ion coordination is a plausible way to
reduce promiscuous activity of metalloenzymes