BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5' GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in E. coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in Genbank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4). Keywords alanine scanning; nicking endonuclease; CatHI; DNA labeling; optical mapping of DNA *Corresponding author Phone: 978-380-7287, Fax: 978-921-1350, xus@neb.com. Individual contributions: P. Zhang expressed wt BspQI and engineered Nt.BspQI and Nb.BspQI; P. Too purified Nb.BspQI, performed nicking assays and produced circular ssDNA by exonuclease III; Siu-Hong Chan cloned and purified CatHI; J. Samuelson constructed a BspQI genomic DNA libraries and performed the methylase selection procedure and "endo-blue" screening; T. Vincze analyzed the 454 sequence contigs; S. Doucette purified wt BspQI; S.Bäckström independently tested Nb.BspQI/exonuclease III on different plasmid constructs.