Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI.
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 A resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a betabetaalpha-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.
Shen
Betty W
BW
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N. A3-025, Seattle, WA 98109, USA.
Heiter
Daniel F
DF
Chan
Siu-Hong
SH
Wang
Hua
H
Xu
Shuang-Yong
SY
Morgan
Richard D
RD
Wilson
Geoffrey G
GG
Stoddard
Barry L
BL
D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D008670:Metals / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics
D008670:Metals; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C068375:endodeoxyribonuclease PacI