Unusual Target Site Disruption by the Rare-Cutting HNH Restriction Endonuclease PacI

• Published in: Structure (London) Vol. 18; no. 6; pp. 734 - 743
• Main Authors: Shen, Betty W., Heiter, Daniel F., Chan, Siu-Hong, Wang, Hua, Xu, Shuang-Yong, Morgan, Richard D., Wilson, Geoffrey G., Stoddard, Barry L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.06.2010
• Subjects: Base Pairing; Base Sequence; Catalytic Domain - genetics; Deoxyribonucleases, Type II Site-Specific; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA Restriction Enzymes - genetics; DNA Restriction Enzymes - metabolism; Metals - chemistry; Protein Structure, Tertiary - genetics; DNA
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2010.03.009

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight-base-pair target recognition sequence 5′-TTAATTAA-3′ has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.