Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate

Abstract

I-TevI is a site-speci®c, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn ®nger, an elongated segment containing a minor groove-binding a-helix, and a helix±turn±helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix±turn± helix subdomain make hydrophobic contacts, the few base-speci®c hydrogen bonds occur in segments that lack secondary structure and¯ank the intron insertion site. The multiple base-speci®c interactions over a long segment of the substrate are consistent with the observed high site speci®city in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases. Keywords: crystal structure/endonuclease/ helix±turn±helix/minor groove/Zn ®nger Introduction Intron-encoded endonucleases are proteins that promote the ®rst step in the mobility of the intron at the DNA level (Belfort and Roberts, 1997). They recognize and cleave an intronless allele of their cognate gene, initiating a replicative gene conversion event that results in the recipient allele also becoming intron-plus. These enzymes are therefore termed homing endonucleases and are grouped into a number of families based on the presence of conserved sequence elements. These are the LAGLIDADG, GIY-YIG, H-N-H and His-Cys box families (Belfort et al., 2001).I-TevI, the group I intron-encoded endonuclease of the td gene of bacteriophage T4, is the best studied member of the GIY-YIG family (Kowalski et al., 1999). The 28 kDa enzyme speci®cally recognizes its lengthy DNA substrate, or homing site, as a monomer ( Figure 1A; Mueller et al., 1995), exhibiting a high degree of sequence tolerance (Bryk et al., 1993). No single nucleotide in the 37 bp target is essential for binding and cleavage, and many multiple substitutions are well tolerated (Bryk et al., 1993. Consistent with this sequence tolerance, ethylation and methylation interference studies indicated that most of the protein±DNA contacts are via the minor groove and the phosphate backbone ( Figure 1B) (Bryk et al., 1993). The primary binding region of the enzyme is~20 bp in length, spanning the intron insertion site (IS), with a second region of contact close to the cleavage site (CS), 23±25 bp upstream of the IS ( Figure 1A). I-TevI demonstrates remarkable¯exibility, recognizing and cleaving homing site derivatives with large deletions (up to 16 bp) and insertions (up to 5 bp) between the IS and CS .The two-domain nature of the homing site is mirrored by the structure of the enzyme ( Figure 1A). I-TevI consists of two functionally distinct domains: an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a long¯exible linker (Derbyshire et al., 1997). The catalytic ...

Zinc finger as distance determinant in the flexible linker of intron endonuclease I- Tev I

“…The recently solved structure of the modular DNA-binding domain of intron endonuclease ITevI complexed with its homing-site duplex (12) has stimulated an analysis of the roles of its individual subdomains. The binding ability of nested sets of deletion derivatives suggests that the H-T-H module at the C terminus, the central minor groovebinding helix, and their joining region are required for DNA binding.…”

“…N-terminal deletions would presumably leave intact most of the phosphate contacts while removing base contacts (Fig. 1C) (12). Qualitatively, the deletion of amino acids 130-167 (Fig.…”

“…A series of deletions was based on contacts found in the cocrystal of the binding domain complexed with its homing-site DNA (12) (Fig. 1 B and C).…”

“…In support of modular evolution, a closely related GIY-YIG homing endonuclease from Bacillus mojavensis, I-BmoI, has two ␣-helical subdomains and an H-T-H subdomain, but no zinc finger (12,31).…”

“…The crystal structure of a complex of the DNAbinding domain, residues 130-245, with a 20-bp DNA duplex has recently been determined (12). Surprisingly, rather than being a compact folded structure, the domain includes three subdomains connected by extended regions (Fig.…”