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Abstract
DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5′ half (CAT) and a canonical 3′ half (GTG). DraIII nicks the 3′ canonical half site at a faster rate than the 5′ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0038-z) contains supplementary material, which is available to authorized users.
Highlights
Restriction endonucleases (REases) are components of restriction-modification systems that occur ubiquitously among prokaryotic organisms and are among the basic tools of molecular biology (Roberts, 2005)
DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand)
A quantitative definition of star activity is provided by the Fidelity Index (FI), which is the ratio of the highest quantity of a REase showing no star activity during digestion to the lowest quantity needed for complete digestion on canonical sites on a specific substrate DNA (Wei et al, 2008)
Summary
Restriction endonucleases (REases) are components of restriction-modification systems that occur ubiquitously among prokaryotic organisms and are among the basic tools of molecular biology (Roberts, 2005). Type II REases are widely used in genetic technologies because of their stable cleavage pattern and are the most well studied They recognize short DNA sequences (3–8 bp) and create double strand breaks at constant positions within or close to that sequence to generate new ends with 5′phosphates and 3′-hydroxyls (Roberts et al, 2003). REases usually cleave specific DNA sequences accurately, certain REases cleave sequences which are similar, but not identical, to their defined recognition sequences under non-optimal conditions, such as the presence of organic solvent, sub-optimal pH values and high enzyme concentrations. This relaxed specificity has been termed star activity. Star activity is not desirable for most REase applications where off-site cleavage is detrimental
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