J . Am. Chem. Sor. 1994, 116. 4085-4086
4085
A Novel Zinc Finger-Based DNA Cutter: Biosynthetic Design and Highly Selective DNA Cleavage
DNA binding domain (Three zmc lingers)
jl
"G~TS..,,.
Makoto Nagaoka. Masaki Hagihara. J u n Kuwahara.' Yukio Sugiura'
and
lnstitute for Chemical Research Kyoto University. Uji, Kyofo 611. Japan Received November 2. 1993 Conversion of a DNA-binding protein to a DNA-cleaving molecule by attachment ofa metal-chelating ligand is one of the most versatile methods for affinity cleaving. These chimeric proteins have largely utilized helix-turn-helixt4 or b-zip'-type motifs and interacted with D N A a s a dimer. Therefore, their targetsitesarelimitedtopalindromicbasesequenceswith a d y a d or a pseudodyad axis. On the other hand, D N A sequences recognized by CysZHisZ-type zinc finger proteins a r e almost asymmetric becauseoftheir monomeric binding mode. We report here the design and function of a new DNA-cleaving metalloprotein consisting of the zinc linger motif. Primary sequence of the zinc finger-based DNA cutter (designated S p l G G H ) comprising two functional domains is shown in Figure I . T h e DNA-binding domain contains the C-terminal region (residues 529-696) oftranscription factor S p l bearing threecontiguous repeatsofthe Cys2His2-typezinc finger motif, which recognizes an asymmetric decanucleotide with consensus sequence 5'-(G/T)GGGCCC(C/A)(G/A)(C/T)-3'.8.9 Each zinc finger domain coordinates a Zn(l1) in a tetrahedral complex. Inaneffort togive DNA-cleavingactivity tozinc finger protein. the tripeptide Gly-Gly-His ( G G H ) was attached t o the N-terminus of the DNA-binding domain because of the availability of a genetic engineering method.I0 The GGH segment originally derived from the copper-binding domain of serum albumin is believed tobind N i ( l l ) ina 1:1 square-planarcomplex with coordination from an imidazole nitrogen, twodeprotonated peptide nitrogens. a n d the terminal amino group, referring to the crystalstructureoftheCulcGGH complex." TheNi(l1)complex
*Author to whom correspondence should be addressed. 'Present address: Faculty of Pharmaceutical Sciences. Tokurhima University. Tokurhima 770. Japan. (1)Chen.C.-H. B.:Sigman. D.S.Scimcr 1987. 237. 1197-1201. (2) BruieqT. W.: Wise. J.: Rorrer. D. S.E.:Sigman. D. S. J. Am. Chrm. V,v
.__.. .._.IW,
I,?
/O.I mM 2-mercaptoethanol)10 timcs and thcn digested with factor Xa a t an cnrymcto-substrateweight ratio of 1:lOO at 25 "C for I h. The resulting SplGGH war purified by reverse-phase Cs HPLC.
*,.GO"
'
0"
L
F.;'sD"
FTi:%;. R
;
;,.I
GERDF
WXKF
1-1:
NIitGG...NCi
...
DNA cleeya e domain
p i p e p r d GGn)
F i g r e 1. Schematic representation of SplCGH. Amino acid residues ofSplGCH are indicated by one-letter abbreviations and numbered from the amino terminus. The tripeptide GGH. the linker region. and the amino acids relevant10 specific base contac116.22 arc represented as bold. outlined. and shadowed types. respectively.
:
i
