LAUSR

Restriction endonucleases (REases) are enzymes that cleave duplex DNA at a recognition site specified by a particular nucleotide sequence. These enzymes are found in a wide range of bacterial species, and serve as a defense against infection by phage. Type IIP REases are typically antiparallel homodimers that recognize a palindromic DNA sequence and cleave within or close to the recognition site. However, some Type IIP REases cleave pseudopalindromic sequences, and some of these may be active as monomers. It is hypothesized that these monomeric REases sequentially cleave the two complementary strands of duplex DNA during a single DNA binding event. Since the two strands of DNA are antiparallel, after cleaving one strand, a monomeric REase would be required to rotate about the axis perpendicular to the DNA to be properly oriented to cleave the second strand. This type of reorientation is known a “flipping”. Although only a small number of proteins have been observed reorienting in this way, protein flipping may play an important role in binding to a specific DNA sequence. Direct observation of flipping during DNA cleavage will greatly improve our understanding of how monomeric REases mediate double strand breaks. We are currently using total internal reflection fluorescence (TIRF) microscopy to observe REase-mediated cleavage of quantum dot-labeled DNA at the single-molecule level. In our highly multiplexed assay, the disappearance of a quantum dot indicates cleavage of the DNA that tethers it to a functionalized glass coverslip. We plan to utilize single-molecule Forster resonance energy transfer (smFRET) to trace individual monomeric REases in real time through the entire process of DNA cleavage from substrate binding to product release. Our observations should provide significant insight into the mechanism by which these enzymes cleave duplex DNA.