The Biochemical Characterization of Binding and Cleavage in HUH-Endonucleases
HUH-endonucleases are diverse enzymes that contain an eponymous motif consisting of a pair of conserved metal-coordinating histidine (H) separated by a bulky hydrophobic residue (U). Replication-initiating HUH-endonucleases (Reps), which are involved in rolling-circle replication in viruses and bacterial plasmids, have been widely explored due to their unique ability to form covalent linkages to the newly exposed 5’ end of cleaved single-stranded DNA (ssDNA) and their minimal sequence specificity requirements. While Reps have been the focus of research in the Gordon Lab, there is still a great diversity of HUH motif-containing proteins to explore, including relaxase (Rlx) enzymes involved in bacterial conjugation and a variety of DNA transposases. Here, we have characterized the cleavage kinetics, substrate binding, sequence specificity/orthogonality of a panel of Reps and Rlxs. Ultimately, these data, along with existing structures, will be used to guide directed engineering and rational design of more specific and orthogonal HUH-endonucleases.