Robert Maunus

DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion

HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G↓CGC in DNA. We report three structures of HinP1I–DNA complexes: in the presence of Ca2+ (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg2+ (post-reactive complex). HinP1I forms a back-to-back dimer with two active sites and two DNA duplexes bound on the outer surfaces of the dimer facing away from each other. The 10 bp DNA duplexes undergo protein-induced distortions exhibiting features of A-, B- and Z-conformations: bending on one side (by intercalation of a phenylalanine side chain into the major groove), base flipping on the other side of the recognition site (by expanding the step rise distance of the local base pair to Z-form) and a local A-form conformation between the two central C:G base pairs of the recognition site (by binding of the N-terminal helix in the minor groove). In the pre- and post-reactive complexes, two metals (Ca2+ or Mg2+) are found in the active site. The enzyme appears to cleave DNA sequentially, hydrolyzing first one DNA strand, as seen in the post-reactive complex in the crystalline state, and then the other, as supported by the observation that, in solution, a nicked DNA intermediate accumulates before linearization.

Structure of HinP1I endonuclease reveals a striking similarity to the monomeric restriction enzyme MspI

HinP1I, a type II restriction endonuclease, recognizes and cleaves a palindromic tetranucleotide sequence (G↓CGC) in double-stranded DNA, producing 2 nt 5′ overhanging ends. Here, we report the structure of HinP1I crystallized as one protein monomer in the crystallographic asymmetric unit. HinP1I displays an elongated shape, with a conserved catalytic core domain containing an active-site motif of SDX18QXK and a putative DNA-binding domain. Without significant sequence homology, HinP1I displays striking structural similarity to MspI, an endonuclease that cleaves a similar palindromic DNA sequence (C↓CGG) and binds to that sequence crystallographically as a monomer. Almost all the structural elements of MspI can be matched in HinP1I, including both the DNA recognition and catalytic elements. Examining the protein–protein interactions in the crystal lattice, HinP1I could be dimerized through two helices located on the opposite side of the protein to the active site, generating a molecule with two active sites and two DNA-binding surfaces opposite one another on the outer surfaces of the dimer. A possible functional link between this unusual dimerization mode and the tetrameric restriction enzymes is discussed.

Cloning and expression of AatII restriction-modification system in Escherichia coli.

The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.