DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target.
R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA, USA.
Betty W
BW
Shen
New England Biolabs, Inc., 240 County Road, Ipswich, MA, USA.
Daniel F
DF
Heiter
New England Biolabs, Inc., 240 County Road, Ipswich, MA, USA.
Keith D
KD
Lunnen
New England Biolabs, Inc., 240 County Road, Ipswich, MA, USA.
Geoffrey G
GG
Wilson
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA, USA.
Barry L
BL
Stoddard
D020856:AT Rich Sequence; D000595:Amino Acid Sequence; D020029:Base Pairing; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D011994:Recombinant Proteins; D004247:DNA; C074767:endodeoxyribonuclease SwaI; D015252:Deoxyribonucleases, Type II Site-Specific; C068375:endodeoxyribonuclease PacI