LAGLIDADG
Homing Endonuclease I-CreI
Christian Hinderer
'10 and Nora Pencheva '09
Contents:
I.
Introduction
Homing endonucleases comprise a diverse family of
proteins that are
found in organisms from all branches of life: eubacteria, archaea, and
eukaryotes. All homing endonucleases are characterized by their ability
to laterally transfer their encoding sequence to a homologous allele
lacking the sequence, a process termed homing.
The homing
endonucleases’ open reading frames (ORFs) are found within
genetically mobile host introns. The intron
elements
carrying the homing ORFs can self-splice at the mRNA level, preventing
any disruption of the host genetic information. Each homing
endonuclease catalyzes the lateral transfer of its ORF by recognizing
and cleaving a specific sequence (known as the homing site)
embedded within a homologous
allele that lacks the endonuclease-encoding intron. Following
endonucleolytic cleavage of the homing site, the homing endonuclease
encoding sequence can be laterally transferred and embedded within the
DNA homing site via two
different routes. Mobile group I
introns utilize homologous
recombination between alleles via a double-strand repair/gene
conversion mechanism. Mobile group II
introns use a reverse
transcription mechanism to synthesize new DNA using the homing
endonuclease
mRNA sequence as a template (Belfort et al., 1995; Jurica et al., 1998;
Chevalier and Stoddard, 2001).
Homing endonucleases are organized in four
general families that include the LAGLIDADG, His-Cys Box,
HNH, and GIY-YIG family. The largest family of endonucleases (with more
than 200 member proteins) is characterized by the presence of a
conserved LAGLIDADG
residues motif (Chevalier and Stoddard, 2001).
The protein
I-CreI is a member of the LAGLIDADG family and it is among the best
biochemically understood homing endonucleases. I-CreI is encoded by a
group
I intron found in the Chlamydomonas
reinardtii chloroplast 23S rRNA
gene (Jurica et al., 1998). I-CreI recognizes a target homing site
that represents a
22-bp long pseudo-palindromic DNA sequence.
Cleavage of the homing site by I-CreI generates two four-nucleotide
long
3’ extended "sticky
ends" .
The cleavage reaction is catalyzed by three divalent cations bound at
and between the enzyme active sites (Chevalier et al., 2001; Chevalier
et al., 2004).
Given their ability of highly selective long-range DNA
recognition and
efficient cleavage, homing endonucleases have emerged as ideal models
for the design of accurate enzymes for DNA cleavage and recombination.
Site-specific mutations of residues in the DNA-binding regions of
homing endonucleases, whose structures are known, have greatly expanded
the range of potential homing targets available (Arnould et al., 2006).
For instance, Redondo
et al. (2008) recently reported two engineered structural derivatives
of I-CreI that were successfully designed to recognize and cleave
target DNA from the human xeroderma pigmentosum group C (XPC) gene.
These findings confirm the utility of homing endonucleases, such as
I-CreI, in providing novel insights into genome engineering and gene
therapy.
II.
General Structure of Endonuclease/DNA
Complex
I-CreI
is a homodimer composed of two
symmetrical monomers.
Each
monomer contains five
alpha helices
lying
on one face of a broad beta ribbon region comprised of four
antiparallel beta strands.
The
association of the alpha and beta regions is
mediated by contacts between four of the helices (a2, a3, a4, a5) and
the internal face of the beta ribbon.
The
external face of the beta ribbon forms the DNA
binding region.
The
fifth helix (a1),
containing the first seven
residues (13-19) of the LAGLIDADG motif (residues 13-21), lies
perpendicular to
the other helices at the interface of the two monomers.
Residues
16 and 17 deviate from the consensus LAGLIDADG
sequence such that in I-CreI Phe-16
and Val-17
replace the
conserved Leu-16 and Ileu-17 in the consensus motif.
Short
range Van der Waals forces mediate the close
interaction between the internal a1 helices of the two monomers.
This
interaction results in the juxtaposition of a
conserved aspartate residue (Asp 20) from each monomer. The two Asp
residues bind three
divalent cations at the protein-DNA interface,
forming the active site of the enzyme near the DNA minor groove.
Conformational
changes in the DNA are induced by I-CreI binding, which
slightly bends the helix
and
compresses the minor groove adjacent to the
enzyme’s active site
(Jurica
et al., 1998; Chevalier et al., 2001;
Chevalier et al., 2003).
III.
DNA Binding
I-CreI interacts
with the DNA homing site through
a network of multiple direct contacts. The residues
mediating DNA binding lie within the beta ribbon region of each I-CreI
monomer. Each monomer utilizes eight residues of this region to
recognize a nine-base pair DNA sequence. The beta ribbons
curve along the
DNA major groove
allowing
for a longer recognition sequence than it is
generally possible with less flexible binding motifs such as a helix-turn-helix
motif The distinctive curvature of the ribbon is dependent
upon the loop
connecting b1 and b2,
which
also contributes to DNA binding through three
residues (Asn-30, Ser-32, and Tyr-33) that hydrogen bond with the DNA
bases T, A, and G, respectively
Sequence-specific
DNA recognition by the beta ribbons
is mediated by residues Arg-70, Gln-44, Arg-68, Gln-26, Lys-28, Gln-38,
Tyr-33, and Ser-32, which project from b1, b2, and b4.
Three
of the most conserved nucleotides in the homing
sequence are those that are recognized by Arg-68, Arg-70 and Gln-38;
each protein residue makes double H-bond contacts with its respective
DNA base.
Nonspecific
contacts to the DNA backbone are made by
residues Lys-48,
Arg-51, Lys-98, Ser-138, and Lys-116.
Indirect
contacts between the DNA minor groove,
separating the homing sequence half sites, and enzyme residues Gln-47
and Asp-20 are made through water-mediated
or metal-coordinated
binding.
(Jurica
et al., 1998; Chevalier et al., 2001;
Chevalier et al., 2003).
IV.
Endonuclease Active Site
When
bound by I-CreI, DNA is bent and the minor groove compressed,
bringing the two scissile
phosphate groups at the active site into close proximity
to each other.
The
catalytic activity of I-CreI requires binding of
three
Mg2+ ions in the enzyme’s active site. In the uncleaved
substrate state, the enzyme is complexed with inactivating Ca2+ ions.
The
most highly conserved residues in the active site,
the C-terminal
aspartate residues (Asp-20 and Asp-20') of the LAGLIDADG
motif,
directly stabilize the catalytic
metal ions at the enzyme-DNA
interface.
Three
other active site residues (Lys 98, Arg 51, and
Gln-47) are also believed to be important for cleavage activity,
though
their role is not fully understood.
The three divalent cations
bound in the enzyme’s active site
form
a line along the minor groove, positioning the
two outer metal
ions against the two scissile
phosphate
groups of each
active site and leaving the third ion directly between the two active
sites of the endonuclease.
This
arrangement allows for the central
metal ion to
participate in the catalytic activity of both of the outer ions.
Each outer metal
ion bound in the I-CreI active site is coordinated by an
octahedral network of six ligands: the oxygen atom of the conserved
Asp-20 residue, the carbonyl oxygen of Gly-19, two non-bridging oxygen
atoms of a scissile
phosphate and a non-scissile phosphate on the
opposite DNA strand, and two water
molecules.
The
central metal ion
is also coordinated in an
octahedral arrangement, with each monomer contributing three ligands:
an oxygen atom from the Asp-20 residue, a 3’ bridging oxygen
atom from a scissile
phosphate, and a non-bridging oxygen from the scissile
phosphate.
(Jurica
et al., 1998; Chevalier et al., 2001;
Chevalier et al., 2003).
V.
DNA Cleavage
DNA
cleavage occurs across the minor groove between the two scissile
phosphate groups. The phosphodiester bond is hydrolyzed by
an
Sn2 mechanism. The HO-
nucleophile is stabilized and
positioned for attack by the outer
Mg2+ ion
adjacent to a scissile
phosphate.
The
3’
oxyanion leaving group is then
stabilized by the internal
Mg2+ ion following DNA strand cleavage.
The
three metal ions remain bound in the cleaved
complex, with the outer
ions now coordinated with a hydroxyl group
attached to the 5’
phosphate rather than a water molecule.
The
resulting double-strand break, consisting of two
four-nucleotide long 3’ cohesive ends, can serve as a trigger
of homologous
recombination that will ultimately transfer the ORF encoding I-CreI to
the cleaved homing DNA site.
(Chevalier
et al., 2001; Chevalier et al.,
2003).
VI.
References
Arnould,
S., Chames,
P., Perez, C.,
Lacroix, E.,
Dulcert, A., Epinat, J.C., Stricher, F., Petit, A.S., Patin, A.,
Guillier, S.,
Rolland, S., Prieto, J., Blanco, F.J., Bravo, J., Montoya, G., Serrano,
L.,
Duchateau, P., Paques, F. 2006. Engineering
of large numbers of highly specific homing endonucleases that induce
recombination on novel DNA targets. J. Mol. Biol.: 355,
443-458.
Belfort,
M.
and Perlman, P.S. 1995. Mechanisms of intron mobility. J.
Biol. Chem.:
270, 30237-30240.
Chevalier,
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2003.
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253-269.
Chevalier,
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uses
three metals, one of which is shared between the two active sites. Nature
Struct. Biol.:8, 312-315.
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B.S.,
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endonuclease.
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Jurica,
M.S., Monnat,
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Redondo,
P., Prieto,
J., Munoz,
I.G., Alibes, A.,
Stricher, F., Serrano, L., Cabaniols, J.P., Daboussi, F., Arnold, S.,
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C., Duchateau, P., Paques, F., Blanco, F.J., Montoya, G. 2008. Molecular
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38, 49-95.
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