DNA nicking by HinP1I endonuclease: bending, base flipping and minor groove expansion.
HinP1I recognizes and cleaves the palindromic tetranucleotide sequence G downward arrowCGC in DNA. We report three structures of HinP1I-DNA complexes: in the presence of Ca(2+) (pre-reactive complex), in the absence of metal ion (binary complex) and in the presence of Mg(2+) (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 (Ca(2+) or Mg(2+)) 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.
Horton
John R
JR
Department of Biochemistry, Emory University School of Medicine 1510 Clifton Road, Atlanta, GA 30322, USA.
Zhang
Xing
X
Maunus
Robert
R
Yang
Zhe
Z
Wilson
Geoffrey G
GG
Roberts
Richard J
RJ
Cheng
Xiaodong
X
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; D002118:Calcium