Hin4II, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: Discovery, cloning and expression in Escherichia coli

The genes encoding restriction-modification system of unknown specificity Hin4II from Haemophilus influenzae RFL4 were cloned in Escherichia coli and sequenced. The Hin4II system comprises three tandemly arranged genes coding for m6A DNA methyltransferase, m5C DNA methyltransferase and restriction endonuclease, respectively. Restriction endonuclease was expressed in E. coli and purified to apparent homogeneity. The DNA recognition sequence and cleavage positions were determined. R.Hin4II recognizes the novel non-palindromic sequence 5′-CCTTC-3′ and cleaves the DNA 6 and 5 nt downstream in the top and bottom strand, respectively. The new prototype restriction endonuclease Hin4II was classified as a potential candidate of HNH nuclease family after comparison against SMART database. An amino acid sequence motif 297H-X14-N-X8-H of Hin4II was proposed as forming a putative catalytic center.