Published online 24 July 2007 Nucleic Acids Research, 2007, Vol. 35, No. 17 5839–5850
doi:10.1093/nar/gkm624
Altered target site specificity variants of the I-PpoI
His-Cys box homing endonuclease
Jennifer L. Eklund1, Umut Y. Ulge3, Jennifer Eastberg3,4 and Raymond J. Monnat, Jr1,2,*
1Department of Genome Sciences, 2Department of Pathology, 3the Molecular and Cellular Biology Program,
University of Washington, Seattle, WA and 4Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Received June 24, 2007; Revised July 27, 2007; Accepted July 30, 2007
ABSTRACT
We used a yeast one-hybrid assay to isolate and
characterize variants of the eukaryotic homing
endonuclease I-PpoI that were able to bind a
mutant, cleavage-resistant I-PpoI target or
‘homing’ site DNA in vivo. Native I-PpoI recognizes
and cleaves a semi-palindromic 15-bp target site
with high specificity in vivo and in vitro. This target
site is present in the 28S or equivalent large subunit
rDNA genes of all eukaryotes. I-PpoI variants able
to bind mutant target site DNA had from 1 to 8
amino acid substitutions in the DNA–protein inter-
face. Biochemical characterization of these proteins
revealed a wide range of site–binding affinities and
site discrimination. One-third of variants were able
to cleave target site DNA, but there was no
systematic relationship between site-binding affinity
and site cleavage. Computational modeling of
several variants provided mechanistic insight into
how amino acid substitutions that contact, or are
adjacent to, specific target site DNA base pairs
determine I-PpoI site-binding affinity and site
discrimination, and may affect cleavage efficiency.
INTRODUCTION
Proteins that bind DNA play a critical role in regulating
gene structure, replication and expression in all organisms.
Biochemical and structural analyses of proteins that bind
specific DNA sequences have begun to provide insight
into the molecular basis of both DNA binding and
sequence-specific DNA recognition (1–3). These analyses
have identified protein folds for DNA binding, together
with a few general rules for the protein-mediated
recognition of specific DNA bases (1,4,5). This work has
also begun to suggest ways to modify the recognition
specificity of existing, sequence-specific DNA-binding
proteins. Two classes of site-specific DNA-binding
proteins that have been the focus for efforts to engineer
new DNA recognition specificities are the Type II
restriction endonucleases (6), and sequence-specific tran-
scription factors (1,7).
The homing endonucleases, a group of highly sequence-
specific DNA-binding proteins, are also being investigated
for recognition specificity engineering. Four different
families of homing endonuclease proteins have been
identified on the basis of protein sequence comparisons,
and one or more families have been identified in all
Kingdoms of life (8,9). The physiologic role of homing
endonucleases is to target the lateral transfer of parasitic
DNA elements known as mobile introns by making a
highly sequence-specific DNA double-strand break in an
intron-less recipient allele (8,9).
The high site specificity of many homing endonucleases
reflects a combination of long (15–40 bp) DNA target or
‘homing’ sites, together with a high degree of sequence
specificity at most target site base-pair positions. A second
intrinsic property of many homing endonucleases is tight
coupling of site recognition to catalysis (10). This is a
particularly attractive feature of homing endonucleases, in
contrast to other potential genome engineering reagents
such as zinc finger nucleases (11). High site specificity
and tight coupling of site binding to catalysis may
reflect the evolutionary history of many homing endonu-
cleases: these two properties in concert permit the
continued lateral transfer—and thus the persistence—of
endonuclease-encoding mobile introns to related target
sites, while minimizing spurious chromosome cleavage
events (12,13).
Our aim was to determine whether structure-guided
protein engineering could be used to alter the DNA
recognition specificity of the eukaryotic homing endonu-
clease I-PpoI, a member of the His-Cys box family of
homing endonucleases. I-PpoI was originally identified as
an open reading frame in a self-splicing mobile intron
found in extrachromosomal copies of the 28S rRNA genes
of Physarum polycephalum, a Plasmodial myxomycete
slime mold (14). It is the best characterized member of the
His-Cys box family of homing endonucleases, one family
Present address:
Jennifer L. Eklund, University of Michigan School of Education, Ann Arbor, MI, USA.
*To whom correspondence should be addressed. Tel: 206 616 7392; Fax: 206 543 3967; Email: monnat@u.washington.edu

2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

in the aab-Me or His-Me endonuclease superfamily
(15,16), and has not thus far been a focus for structure-
guided design.
The active form of I-PpoI is a 36 kDa homodimer that
cleaves a 15-bp semi-palindromic DNA target site in the
28S Physarum rDNA locus, and in the corresponding
large subunit rRNA genes of all eukaryotes [(17–19);
Figure 1]. Rare target sites may also exist outside the
rDNA repeats (20). We had previously determined high-
resolution apo- and co-crystal structures of native and
mutant forms of I-PpoI that allowed us to identify the
molecular basis for high affinity site binding and cleavage
(21,22). We had also identified amino acid changes that
interfere with I-PpoI catalysis or site binding, together
with DNA base-pair changes that disrupted target site
cleavage by native I-PpoI protein (23–26). These data were
used to construct a yeast one-hybrid (Y1H) screening
assay to identify I-PpoI protein variants able to bind a
specific mutant target site in vivo. Biochemical character-
ization together with computational modeling were used
to gain insight into the molecular basis for mutant site
recognition and cleavage by variant I-PpoI proteins.
MATERIALS AND METHODS
Plasmids and yeast strains
Yeast reporter plasmids were constructed using the YEp24
two-micron plasmid vector or the integrating plasmid
vector pRS404 (27,28). An I-PpoI-specific YEp24-lacZ
reporter was constructed by inserting I-PpoI target sites
into the SalI site upstream of a cyc5 promoter and lacZ
gene. Target site inserts were prepared by annealing
phosphorylated oligonucleotides (PPOSITES5 and
PPOSITES6; all oligonucleotide sequences are given in
Supplementary Data Table 1), or oligonucleotides that
when annealed created three oriented copies of native or
mutant I-PpoI target sites (oligonucleotides PPOX3_WT
and _6 #1-4). Yep24-HIS3Ppo was constructed from the
resulting plasmids by replacing the lacZ gene with a PCR
fragment containing the budding yeast HIS3 gene.
Figure 1. Amino acid geometries were determined from the I-PpoI co-crystal structure (21). (A) Stereo representation of the native I-PpoI
DNA–protein interface. Canonical contacts are present between Q63:
6A and R74:
7G. The native
6A/T base pair is shown in yellow. Amino
acid geometries were modeled using RosettaDesign. Sequence-specific hydrogen bonds are shown as black dotted lines. (B) Sequence of the native
I-PpoI target or ‘homing’ site. Site cleavage across the minor groove (staggered line) generates complementary 4 base, 30 OH-extended single-stranded
ends. The convention for numbering target site base-pair positions is shown above the top strand. (C) Sequence and cleavage sensitivity of native and
five variant I-PpoI target sites with palindromic base substitutions (shown lower case bold) at positions
3 to
7 assayed for native I-PpoI cleavage
sensitivity. Cleavage sensitivity is indicated to the right of each sequence (‘+++’, fully sensitive; ‘–’, no cleavage). (D) Representative cleavage
assays of native and mutant I-PpoI target sites by native I-PpoI protein. Linearized plasmid DNA (10 mM) was digested for 1 h with 10 pM to 1 mM
native I-PpoI prior to agarose gel electrophoresis. (‘uncut’, plasmid substrates; ‘cut’, site-specific cleavage products).
5840 Nucleic Acids Research, 2007, Vol. 35, No. 17

An integrating version of Yep24-HIS3Ppo was con-
structed by transferring an EcoRI fragment containing a
multiple-cloning site, minimal promoter and HIS3 gene
into the yeast TRP1 integration vector pRS404, and then
inserting three copies of the native or +6C/–6G mutant
I-PpoI target site as described earlier Yeast containing an
integrated copy of pRS404-HIS3Ppo were constructed by
transforming MfeI-cleaved linear plasmid DNA into yeast
strain W1588-4C (MATa ade2-1 can1-100 his3-11, 15
leu2-3, 112trp1-1 ura3-1 RAD5), followed by selection on
SD media lacking tryptophan (29,30). Selection for HIS3
reporter expression was performed by growth on SD
medium lacking histidine, followed by a demonstration of
growth suppression by 3mM 3-aminotriazole. All reporter
plasmids and strains were verified by DNA sequencing of
the I-PpoI target site region.
Generation of I-PpoI protein variants
CR library. Expression plasmids for Ppo-activation
domain (AD) fusion proteins were constructed by insert-
ing I-PpoI open reading frame cassettes in-frame into the
GAL4 activation domain plasmid pOAD (kindly provided
by Stan Fields, University of WA, USA). The corrected
map and sequence of this plasmid can be found at:
depts.washington.edu/sfields/protocols/pOAD.html. After
removing flanking BamHI sites, the open reading frame of
catalytically inactive H98A I-PpoI was inserted into the
NcoI and PstI sites of pOAD to form pOAD-Ppo. A 3 kb
insert or ‘dummy’ fragment was inserted into the NdeI–
BamHI site in the I-PpoI ORF to create pOAD-Ppo+d.
This ‘dummy’ fragment was subsequently replaced with an
oligonucleotide insert encoding 25 codons of the I-PpoI
ORF to restore the open reading frame and randomize
I-PpoI residues N57, R61, Q63, K65 and R74. This insert
was constructed by the annealing of three oligonucleo-
tides, PPO(-)5, PPO+ NDEI and PPO NDEI SPLINT
and ligation, at 500-fold molar excess, into NdeI-cut
pOAD-Ppo+d. The PPO(-)5 oligonucleotide region con-
taining randomized codons was converted to double-
stranded DNA by primer extension with Klenow DNA
polymerase prior to cleaving the resulting insert and
plasmid with BamHI (31), ligation and electroporation
into E. coli TB1 cells. The number of independent
transformants was determined by plating a small portion
of the pooled transformation, and the library was
amplified by growth overnight in 1 l of L-broth followed
by plasmid DNA isolation [(32); Qiagen MegaPrep].
RD1 and RD2 libraries. The RD1 and RD2 libraries were
constructed by replacing the region encompassing I-PpoI
residues 55–76 with an insert generated by PCR-mediated
assembly of degenerate oligonucleotides. Five oligonu-
cleotides (the 4 +6G_B oligonucleotides and PPOOADN
were used to assemble the N-terminal portion of the
I-PpoI H98A ORF. Two +6G_E oligonucleotides and
PPOOADC (RD1) or PPOOADCdeg (RD2) were used to
assemble the C-terminal portion of the I-PpoI ORF.
PPOADCdeg included a 9 bp randomized ‘molecular bar
tag’ sequence to allow different starting plasmids to be
distinguished. Both PCR assembly products were gel
purified by polyacrylamide gel electrophoresis (PAGE).
The N-terminal assembly product was extended in a
second PCR reaction using a pool of 27 (RD1) or 18
(RD2) +6_M oligonucleotides together with PPOOADN.
The resulting product was gel-purified and amplified
with recombination cloning primers AD70F and AD70R
(kindly provided by Stan Fields, University of WA; http://
depts.washington.edu/sfields/protocols/protocols.html) to
generate a full-length I-PpoI open reading frame insert,
and then used for in vivo gap repair of PvuII + NcoI-
cleaved pOAD-Ppo plasmid DNA.
Ppo-AD libraries were characterized by sequencing
plasmid DNA isolated from independent colonies.
Seventy-four CR library plasmids were sequenced, 11
from plasmid preps and 63 from PCR products generated
using PADSCREENF and PADSCREENB oligonucleo-
tides. RD1 and RD2 were characterized by sequencing
yeast colony PCR products. PCR and the sequencing were
performed with PADSEQF and 882 oligonucleotide
primers. The resulting forward and reverse sequences
were aligned using Sequencher (www.sequencher.com).
Site-directed mutagenesis. The I-PpoI open reading frame
was subcloned from Ppo-AD fusion plasmids into a
pET11c derivative containing an N-terminal hexa-
histidine affinity purification tag prior to expression in
E. coli and purification by Ni-NTA affinity chromato-
graphy (Qiagen). The pET11c-histagPpo expression vector
was constructed by annealing oligonucleotides pET-
histagf and pET-histagr containing the affinity tag,
followed by ligation into the NdeI site of pET-Ppo (23),
to generate pET11c-histagPpo. Site-directed mutagenesis
using a QuikChange protocol (Stratagene) was then used
to generate pET vectors expressing variants A, B, C, F, P,
Q, R, S, T, Y and H98A/L116A. Variants U, V and W
were made by site-directed mutagenesis of the variant C
expression vector. Variants D, E, G, H, J, K, L, M and
N were constructed by inserting an NcoI–BamHI frag-
ment from RD2 Ppo-AD plasmids into the open reading
frame of pET11c-histagPpo.
Library screening and b-galactosidase activity assays
CR or RD library screening was performed by trans-
forming CR or RD plasmid libraries, together with the
+6C/-6G lacZ reporter plasmid Yep24-lacZPpo, into
cells possessing an integrated copy of the pRS404-
HIS3Ppo reporter plasmid. The resulting cells were then
selected for growth on histidine-minus media followed by
a visual screen for b-galactosidase activity. The number of
independent co-transformants was determined by plating
a small known fraction of the transformed yeast on
SD–leucine–uracil plates. Library screening was per-
formed by dense plating of transformed cells on
SD–leucine–uracil–histidine plates supplemented with
3mM 3-aminotriazole, followed by growth at 308C for
7 days. Large colonies were then picked into 1ml 96-well
deep blocks containing SD–leucine–uracil media and
grown for 7 days without shaking at 308C.
A colorometric screen for b-galactosidase reporter gene
induction was used to identify Ppo-AD plasmids with
Nucleic Acids Research, 2007, Vol. 35, No. 17 5841

in vivo site-binding activity. In brief, cells were pelleted,
then resuspended and permeabilized in Z-buffer using
SDS and chloroform (33). The b-galactosidase substrate
ONPG was added to 0.6mg/ml, and wells were incubated
at 308C to monitor color change. Incubations were
stopped between 2 and 4 h by adding Na2CO3 prior to
visual screening to identify positive wells by comparison
against positive control (H98A Ppo-AD and native I-PpoI
site lacZ reporter) or negative control (H98A Ppo-AD and
mutant +6C/–6G site lacZ reporter) cells.
Ppo-AD coding plasmids were recovered from activity-
positive cells by use of either a Qiagen or Sigma miniprep
kit, amplified by transformation and growth in E. coli,
and then transformed into yeast strain W1588-4C. We
assayed at least eight transformants from each original
positive colony for b-galactosidase induction by ONPG
assay prior to streaking cells on SD–leucine plates
supplemented with 5-fluoroorotic acid (5-FOA) to elim-
inate the URA3 reporter plasmid. In order to verify site-
specific b-galactosidase induction, plasmid DNAs from
activity-positive cultures were retransformed as described
above with a +6C/6G or native site reporter plasmid.
The DNA sequence of the Ppo-AD open reading frame
was determined from the same plasmid preparation by
sequencing using a PADSCREENF sequencing primer.
Quantitative b-galactosidase activity assays were per-
formed to further characterize several I-PpoI variants by
using a modification of the screening assay as described
above (33). Cells were grown overnight at 308C in
SD–leucine–uracil media. Cell density was determined by
measuring absorbance at 600 nm, and 2ml of each culture
was pelleted and resuspended in 0.8ml of Z-buffer
followed by the addition of 50 ml 0.1% SDS and 50 ml
chloroform with vortexing for 30 s. Activity was measured
by adding 160 ml of 4mg/ml ONPG in Z-buffer followed
by incubation at 308C, and stopped by adding 0.4ml
Na2CO3 after the development of yellow color. Cell debris
was spun out of reactions prior to measuring absorbance
at 420 nm and calculating units of b-galactosidase activity.
All reported values represent an average of three
independent determinations.
Protein expression and purification
Variant I-PpoI proteins subcloned into pET11c-histagPpo
were expressed in E. coli host strain BL21(DE3)
(Novagen). Single colonies were picked into 2–3ml of
L-broth supplemented with 100 mg/ml carbenicillin and
0.2% glucose, then grown overnight at 378C. Overnight
cultures (300ml each) were then diluted into 100ml of
L-broth supplemented with carbenicillin and 1mM zinc
acetate and grown for 2–4 h at 378C. Protein production
was induced by adding 1mM IPTG. After induction
for 3–5 h at 378C, cells were pelleted and stored overnight
at 808C.
Protein was purified from frozen cell pellets by thawing
and resuspending cells in 1ml of lysis buffer (50mM
NaH2PO4, 300mM NaCl and 10mM imidazole) contain-
ing 1mg/ml lysozyme on ice, followed by the addition of
protease inhibitors (1mM PMSF, 1 mg/ml leupeptin and
1 mg/ml pepstatin) and sonication. RNAse A (10mg/ml)
and DNAse I (5mg/ml) were added to cell lysates followed
by incubation on ice for 15min. Cell debris was pelleted at
48C, and Ni-NTA resin (100 ml; Qiagen) was added to the
cleared supernatant prior to incubation at 48C on a roller
for 1–2 h. Resin was pelleted from the supernatant in
a benchtop centrifuge at 48C, washed once with 1ml of
20mM imidazole buffer (50mM NaH2PO4, 300mM NaCl
and 20mM imidazole), and then washed twice with 1ml
50mM imidazole buffer (50mM NaH2PO4, 300mM NaCl
and 50mM imidazole). Protein was eluted with 3 50 ml
washes of 50mM NaH2PO4, 300mM NaCl and 250mM
imidazole. Eluates were pooled to determine protein
concentration by Bradford assay prior to the addition
of glycerol to 50% (v/v) and storage at 208C.
Polyacrylamide gel electrophoresis of protein samples
followed by Coommassie staining indicated that I-PpoI
protein preparations were typically >75% pure with no
other major contaminating bands.
Site-binding affinity and cleavage assays
The binding affinity (Kd) and cleavage of I-PpoI target
sites were determined as previously described (26). The
substrates for binding affinity assays were formed by
annealing gel-purified PPOx1 oligonucleotide pairs in
which (+) strand oligonucleotides had been end-labeled
with 32P. Kd values were determined from a minimum of
two, or in most cases three, independent assays. Cleavage
assays were performed using linearized plasmids contain-
ing either three copies of native or mutant I-PpoI target
sites inserted into the SalI site of pRS404-HIS3Ppo, or
single site PPOx1_Sal oligonucleotides pairs that had been
annealed and inserted into pBlueScriptII KS(+) plasmid
DNA (Stratagene). pRS404 plasmids were linearized with
SnaBI, and pBluescriptII plasmids with XmnI, prior to
performing cleavage assays.
Computational modeling of I-PpoI variants
Modeling of the I-PpoI DNA interface of native and
variant proteins was done using RosettaDesign (34,35).
In brief, changes to the substrate DNA sequence and the
amino acid sequence of the protein were simulated in
silico, and the DNA–protein complex was allowed to relax
according to an energy function that mimics protein
folding. I-PpoI variants E, G and T were modeled and
visualized for inspection using the PyMOL molecular
viewer (DeLano Scientific LLC).
RESULTS
Cleavage sensitivity of I-PpoI target site variants
We identified I-PpoI target sites that were cleavage-
resistant when challenged with native I-PpoI protein. We
focused initially on 5-bp positions,
3 to
7, and on base-
pair substitutions at these sites that were known to affect
cleavage efficiency on the basis of previous homing site
degeneracy, biochemical or functional analyses (24,25,36).
These base-pair positions are in a well-ordered part of the
I-PpoI DNA–protein interface (Figure 1A). Palindromic

3G>A/C>T and
4G>C/T>G base-pair substitutions
5842 Nucleic Acids Research, 2007, Vol. 35, No. 17

were permissive of cleavage only at high protein concen-
trations (10 nM for the
3 position; 1 mM for the
4
position). In contrast, base-pair substitutions at positions

5 (T>A/C>T),
6 (A>C/T>G) and
7 (G>A/C>T)
completely inhibited cleavage at all protein concentrations
tested (Figure 1D; data not shown). Of the six possible
base-pair changes from the native +6A/6T target site,
+6A>C/6T>G substitutions most strongly inhibited
site cleavage by native I-PpoI (Figure 1C and D; data not
shown). We chose this target site for re-engineering of the
site recognition specificity of I-PpoI.
A yeast one-hybrid screening assay
A yeast one-hybrid (Y1H) screen was used to identify
I-PpoI protein variants that bound the +6C/6G target
site in vivo to induce reporter gene expression. To establish
this assay, we fused a Saccharomyces cerevisiae Gal4p
transcriptional activation domain to the N-terminus of
catalytically inactive H98A I-PpoI to generate Ppo-AD.
The H98A substitution was incorporated to abolish the
catalytic activity of I-PpoI without disrupting high affinity
site binding [Figure 2A; (22,23,26)]. A Ppo-AD negative
control protein was constructed by fusing L116A I-PpoI
to the Gal4p activation domain. L116A I-PpoI is
catalytically inactive, and does not bind I-PpoI target
site DNA (26). Reporter activity was determined using a
budding yeast HIS3 or plasmid-borne bacterial lacZ
reporter genes located downstream of 1 or more I-PpoI
target site and a minimal promoter (Figure 2B). Site
binding was quantified by the induction of b-galactosidase
activity, and was strongest when the lacZ reporter gene
was flanked by 6 direct repeats of the native I-PpoI target
site: 25-to-75-fold above the ‘no site’ or negative control
background. The Gal4-L116A Ppo-AD negative control
protein did not induce reporter expression above back-
ground on any target site or number of repeats (Figure 2C;
data not shown). We performed all subsequent library
screens using reporter genes flanked by three target sites,
as these reporters could be readily constructed and gave
reporter activity well above background.
Generation and screening of I-PpoI protein variant libraries
In order to generate protein variants to screen against
the mutant +6C/6G target site, we made substitutions
in the I-PpoI DNA–protein interface at residues that
contacted base–pair position 6 or adjacent base–pair
positions. This ‘contacting residue’ (CR) library was
designed to allow all 20 amino acid residues at positions
N57, R61, Q63, K65 and R74, and had a maximum
potential complexity of 3.2 106 (205) protein variants.
The resulting CR library was difficult to construct, and
thus not large enough to encompass all predicted variants.
Upon experimental verification, the CR library was found
to encode 1.6 105 different protein variants that each
had an average of 4.7 amino acid substitutions
(CR library, Table 1).
The CR library was screened by Y1H in two steps.
We isolated yeast transformants that grew on histidine-
minus plates containing 3-aminotriazole (3-AT), and then
determined whether the same Ppo-AD plasmids could
induce in vivo b-galactosidase expression. The Ppo-AD
open reading frame of active variants was then sequenced
to identify amino acid substitution(s) that conferred in vivo
activity. Among the 2 106 colony transformants and
4600 His+ colonies screened, only one CR library
transformant had in vivo activity on both reporter genes.
This variant (variant A, Table 2) had four amino acid
substitutions at residue positions randomized during CR
library construction.
Variant A served as a starting point for the construction
of two additional, rationally designed (RD) libraries that
were used to further explore determinants of I-PpoI
recognition and in vivo activity on native and +6C/6G
target sites. The first RD library allowed +6C/6G
contacting residue 63 to vary among seven amino acid
residues: the native Q, or D, E, K, N, R or Y. Neighboring
b-strand residues A55, N57, W62, Y64, R74 and G76 were
substituted with residues that differed slightly in size and/
or polarity from native residues found at these positions
(RD1; Table 1). The predicted maximum complexity of
the RD1 library was 3.5 104. Sequencing indicated that
Figure 2. Yeast one-hybrid assay used to identify GAL4-I-PpoI fusion
proteins that bind target site DNA in vivo. (A) GAL4-I-PpoI fusion
protein generated by inserting the open reading frame of H98A I-PpoI
downstream of the GAL4 transcriptional transactivation domain
(GAL4-AD). The H98A substitution abolishes I-PpoI catalytic activity
without affecting site-binding affinity. (B) Reporter genes to detect
in vivo binding of GAL4-I-PpoI were constructed in the yeast plasmid
YEp24 or in the TRP1 integration vector pRS404 by inserting three
direct repeats of native or mutant I-PpoI target site upstream of a
minimal promoter and the budding yeast HIS3 or bacterial lacZ gene.
(C) In vivo activity of H98A (positive control) and L116A (negative
control) GAL4-I-PpoI fusion proteins in budding yeast, on native or
+6C/-6G target site b-galactosidase reporter plasmids. Activity is
measured by a quantitative b-galactosidase activity assay (see Methods
section).
Nucleic Acids Research, 2007, Vol. 35, No. 17 5843

72% of RD1 Ppo-AD plasmids had intact open reading
frames. Thus the completed RD1 library of 1.8 105
members was sufficiently large to include all RD1 design
variants (RD1, Table 1).
In vivo screening of RD1 against +6C/6G target site
reporter genes yielded 352 His+ colonies, and 15 plasmids
from this pool were verified by re-transformation and
quantitative lacZ activity prior to sequencing. Five active
Ppo-AD plasmids had the same Ppo-AD amino acid
substitutions found in variant A (Table 2). Six different
RD1 starting plasmids harbored the same Q63R and
K65R substitutions (Table 2, variant E), as indicated by
plasmid DNA sequence differences. Four other plasmids
had the variant E Q63R and K65R substitutions together
with 2 to 4 other substitutions at residues targeted during
library construction (variants B, C, D and F; Table 2).
A second rationally designed library was constructed to
determine whether additional residue substitutions could
augment the in vivo activity of Ppo-AD proteins identified
in the CR and RD1 libraries. The RD2 library design
features were: (i) three positions were fixed as residues
found in active variants of RD1 (63R, 64Y and 76G);
(ii) residue 55 was an A, G, L or V, and residue 62 was an
F or W, again as previously identified in active variants;
(iii) contacting residues 57, 61, 65 and 74 were varied
Table 2. Biochemical properties and residue substitutions in I-PpoI variants
Variant Source Binding Kd (nM) Site cleavage Residue at position
WT +6C/6G WT +6C/6G 55 57 61 62 63 64 65 74 76
H98A SDM 0.11
0.02 1000 ++++ No A N R W Q Y K R G
L116A SDM 350
60 1000 No No
T SDM 0.034
0.01 6
1.5 ++++ +/ R
B RD1 110
10 130
30 No No G F R R T
U SDM 130
40 1000 +++ No V C R T
Y SDM 180
60 1000 + No C R R
D RD1, RD2 230
40 240
80 No No L R R
E RD1, RD2 280
90 140
40 ++ No R R
H RD2 460
140 310
90 No No F R R
P SDM 480
30 570
150 ++++ No V
R SDM 500
20 1000 No No K
Q SDM 620
90 570
160 ++ No A
W SDM 620
160 480
100 No No V C R R
A CR, RD1 630
190 1000 No No C R R T
J RD2 760
40 430
50 No No I R R
V SDM 810
120 540
140 No No V C R T
S SDM 820
170 1000 ++ No R
G RD2 1000 190
40 No No K R R
N RD2 1000 300
10 No No V K R R
C RD1 1000 440
80 No No V C R R T
L RD2 1000 500
100 No No L K R R
M RD2 1000 1000 No No L F R R
K RD2 1000 1000 No No L K F R R
F RD1 1000 1000 No No I C I R W R T I
The designation and source of each I-PpoI variant protein are listed in the first two columns. Positive (H98A) and negative (L116A) control proteins
are listed in the first two rows for comparison. Binding affinities for native and +6C/–6G target site DNAs, determined by gel shift assay, are given
in columns 3 and 4 and are arranged in order of decreasing affinity for the native I-PpoI target site. The ability of each I-PpoI variant to cleave
native or +6C/–6G target site DNA was determined after reverting the catalytically inactivating H98A substitution that was required to establish the
yeast one-hybrid assay. The final columns detail amino acid substitutions at nine positions in the DNA-protein interface. A blank box indicates
presence of the native residue (shown top in single letter code). Key: CR, contacting residue library; RD1, RD2, rationally designed protein variant
libraries RD1 and RD2; SDM, site-directed mutagenesis. Note: This Table has been reformatted in Supplementary table 2 to show variants
rank-ordered on the basis of increasing number of residue substitutions at nine positions in the DNA–protein interface.
Table 1. Residue substitutions and library statistics for three I-PpoI
yeast one hybrid libraries and screens
Residue I-Ppol variant library
CR RD1 RD2
A55 A,G,V,L,I A,G,V,L
N57 All N,C Y,E,D,R,K,Q,N,C
R61 All Y,E,D,R,K,Q,N,C
W62 W,F,L,I,Y F,W
Q63 All Y,E,D,R,K,Q,N R
Y64 Y,F,S,T,W
K65 All K,R Y,E,D,R,K,Q,N,C
R74 All R,T Y,E,D,R,K,Q,N,C
G76 A,G,V,L,I
Maximum
complexity
3.2 106 3.5 104 3.2 104
Library size 1.6 105 1.8 105 1.4 105
Colonies
His-screened
2 106 2.5 105 2.1 105
His+lacZ+
plasmids recovered
1 6 9
The nine I-PpoI amino acid residues targeted for substitution during
library constructions are indicated in the left column, together with
substitutions made at each residue in the contacting residue (CR) or
two rationally designed (RD1 and RD2) protein variant libraries.
Maximum complexity is the predicted complexity of the library.
Library size is the experimental complexity of the library, determined
from the number of independent colonies that contained inact open
reading frames.
5844 Nucleic Acids Research, 2007, Vol. 35, No. 17

among eight amino acid residues commonly found in
homing endonuclease DNA–protein interfaces (Y, E, D,
R, K, Q, N, C; Table 1, RD2) and (iv) inclusion of a 9 bp
post-C-terminal ‘molecular bar code’ to allow different
RD2 plasmid molecules to be identified and distinguished.
The RD2 library had a predicted maximum complexity of
3.2 104. Sequencing indicated that 67% of RD2 library
plasmids had intact Ppo-AD open reading frames. Thus
the completed RD2 library of 1.4 105 members was
sufficiently large to include all designed RD2 variants.
Thirty-five RD2 plasmids displayed in vivo activity
against +6C/–6G reporter genes. These plasmids encoded
nine different Ppo-AD proteins: seven Ppo-AD variants
were unique to the RD2 library (variants G, H, J, K, L, M
and N), whereas two variants had been previously
identified in RD1 (variants D and E; Table 2). Ppo-AD
variants D, H, K and L were independently isolated, from
3 to 6 times each from RD2, as indicated by plasmid
molecular bar code sequences. In addition to these library
variants, we constructed nine I-PpoI variants (variants P
to Y, Table 2) by site-directed mutagenesis. This was done
to determine the contribution of specific amino acid
residues identified in the CR, RD1 and RD2 libraries to
target site binding and cleavage. We reasoned that specific
residue substitutions in library isolates might promote or
interfere with site binding and cleavage, and thus wanted
to assay individual substitutions in defined sequence
contexts. These site-directed mutants were generated, in
consequence, in either the native or variant A or C I-PpoI
sequence contexts.
Biochemical characterization of I-PpoI variant proteins
In order to characterize the site-binding properties of
Ppo-AD variants, we expressed and purified variant
proteins from E. coli and then determined their dissocia-
tion constants (Kd’s) on native and on +6C/–6G target
site DNAs. We also determined the ability of selected
variants to induce reporter gene expression in vivo to allow
a comparison of in vitro and in vivo site-binding properties
(Figure 3). The I-PpoI portion of each Ppo-AD fusion
protein was subcloned into a pET11c expression vector
containing an N-terminal six-histidine tag to facilitate
purification. In order to assay site cleavage as well as
binding by specific variant proteins, we used site-directed
mutagenesis to revert the catalytically inactivating H98A
substitution required to establish our Y1H assay.
Dissociation constants for variant proteins were deter-
mined by electrophoretic gel mobility shift analysis using
+6C/6G and native target site DNAs. Fourteen of 22
variants displayed a higher affinity for +6C/6G homing
site DNA than did native I-PpoI, although this site-
binding preference was modest (typically <5-fold;
Table 2). Seven of the remaining variants displayed no
site-binding preference. One variant, T, displayed high
binding affinity for both the native and +6C/6G target
sites and a 175-fold higher affinity for native target site
DNA (Table 2). We also determined the binding affinity of
native I-PpoI and four variant proteins on target site
DNAs with base-pair substitutions at the
6 position, the

7 position and in one instance
6/
7 position
substitutions. The variants had amino acid substitutions
at position 63 contacting the
6 bp; at residue 74
contacting the
7 bp; and at residues 55, 57 and 65 in
adjacent regions of the DNA–protein interface. None of
these variant proteins bound any mutant target site DNA
with high site affinity or specificity (Table 2, and
Supplementary Table 2).
Seven of 22 variant I-PpoI proteins cleaved native
homing site DNA in vitro. Cleavage activity was
determined on linear plasmid substrates that contained a
single I-PpoI target site. Both native I-PpoI and variant T
bound and cleaved native target site DNA, though did not
cleave +6C/–6G target site DNA in the presence of Mg2+
or Mn2+ (Figure 3A; data not shown). Other variant
proteins with activity cleaved only native target site DNA
(variants E, P, Q, S, U and Y; Table 2, Figure 3A).
In order to better understand the relationship between
biochemical properties and in vivo activity of variant
proteins, we quantified the ability of six variants to induce
a lacZ reporter gene in budding yeast. Variants A, C, E,
G, J and T were chosen for analysis as they represent a
range of in vitro binding affinities. Expression plasmids for
each variant as a Ppo-AD fusion were transformed into
yeast containing a native or +6C/–6G target site LacZ
reporter gene plasmid. b-Galactosidase activity was
quantified for three independent colonies grown in liquid
media and assayed on the same day. Protein variants C, G
and J had higher activity on +6C/–6G than on native
target site reporters. This paralleled the 1.8-to-5.3-fold
higher binding affinity of these variant proteins for +6C/–
6G sites (Table 2 and Figure 3B). A graphical summary of
in vitro site binding and site cleavage analyses, for all
variants, is shown in Figure 3 (panels C and D).
Computational modeling and analysis of I-PpoI protein
variants
Structural modeling of base pair and residue substitutions
in the I-PpoI DNA–protein interface was used to gain
mechanistic insight into the properties of several I-PpoI
variant proteins. When I-PpoI is bound to native target
site DNA, Q63 makes an energetically favorable, gluta-
mine:adenine contact with two hydrogen bonds to adenine
6. R74 makes a canonical contact with guanine 7. K65
makes an H-bond contact to guanine 9, whereas R61
makes a series of backbone contacts between base pairs
3 and 4 (Figure 1A). Modeling indicates the +6C/–6G
base-pair substitution inhibits site binding and cleavage by
disrupting the glutamine:adenine 6 contact, and by forcing
the Q63 residue to rotate 1358 into and in part disrupt the
adjacent R74:guanine 7 bp contact (Figure 4A and B).
Several residue substitutions found in variant G could
in part restore mutant +6C/–6G target site binding. These
were Q63R at the +6C/–6G base-pair-contacting position
(present in 17 variants) K65R (present in 17 variants)
and R61K (present in 4 variants; Table 2 and Figure 4,
compare panels B and C). Modeling indicates that Q63R
substitutions can in part restore mutant site-binding
affinity by making a new contact at base-pair position 5,
and by restoring an R74:guanine 7 canonical base-pair
contact (Figure 4C). Modeling also provides an
Nucleic Acids Research, 2007, Vol. 35, No. 17 5845

explanation for why selected variants with Q63R sub-
stitutions, e.g. variant E, show preferential binding of the
mutant +6C/6G target site: when R63 packs in the
native DNA interface, it leaves a small cavity adjacent to
the native
6A/T base pairs. This cavity is partially filled
by the cytosine in the +6C/–6G substrate (Figures 1A and
4B; data not shown). These results indicate that the native
contacting residue, Q63, acts as a gatekeeper at the

6A:T base pair by controlling contacts at this and
adjacent base-pair positions.
Variant T, which contains a K65R substitution, showed
a marked increase in binding affinity for both native and
+6C/–6C target sites. Modeling of this substitution
revealed that K65 forms a single H-bond to the guanine
base at base pair 9, whereas K65R substitution make two
H-bonds to base pairs at positions 8 and 9 (compare
Figure 4 panels B and C). These contacts are energetically
more favorable than the single contact made by the native
K65 residue (1.22 units for R65 versus 1.09 units for
K65; RosettaDesign analyses not shown). Neither K nor
R65 residues contact target site position 6, and thus do not
discriminate between native or mutant +6C/–6G target
site DNAs.
R61K substitutions decrease the DNA-binding affinity
of I-PpoI, while increasing target site selectivity to favor
the binding of mutant, +6C/–6G target site DNA
Figure 3. Target site cleavage and site binding by I-PpoI variant proteins. (A) In vitro cleavage of native and +6C/–6G target site DNAs by native
and 8 variant I-PpoI proteins. Plasmid DNA (10 nM) containing a native (WT, top panel) or +6C/–6G variant (bottom panel) target site was cut
with XmnI to generate a 3 kb linear DNA substrate (‘uncut’) that was cleaved with 0.1 mM native (WT lanes) or variant I-PpoI protein (rightmost 8
lanes, Y to C) for 30min at 378C. I-PpoI target site-specific cleavage generated 2 and 1 kb product fragments (‘cut’). M=1kb DNA size marker
(New England Biolabs). Controls include plasmid cleaved with XmnI alone (– lanes) or plasmid cleaved with XmnI, and with AflII that cleaves the
central 6 bp of the I-PpoI target site (+ lanes). (B) In vivo yeast one-hybrid reporter gene activity of selected I-PpoI protein variants. LacZ reporter
gene activity was determined by expressing specific proteins in yeast in the presence of native (site W) or +6C/–6G (site 6) I-PpoI target site reporter
plasmids, then measuring b-galactosidase activity in permeabilized cells using ONPG as a substrate. Proteins included as controls were H98A Ppo-
AD (98A; positive control) and L116A (116A; negative control). Variant T was assayed independently of the other five variants shown, and thus is
displayed with a simultaneously performed H98A control (rightmost 4 bars). All activities represent the mean
SD of three independent colonies
grown and assayed on the same day. (C and D) Graphical summary of I-PpoI variant protein site binding and cleavage properties. Site-binding
affinities (nM) for native (X axis) or +6C/–6G (Y axis) target site DNAs. Variant proteins with partial or full cleavage activity on native homing site
DNA are indicated, respectively, by circled X’s or filled circles. Only one I-PpoI variant protein, T, had any detectable cleavage activity on +6C/–6G
target site DNA. The identities of the variants in the dashed line box in the upper right of panel C are shown in expanded panel D.
5846 Nucleic Acids Research, 2007, Vol. 35, No. 17

(compare, e.g. variants E and G, Table 2). Modeling
indicates that the R61K substitution modifies back-
bone contacts in the native interface between base pair
positions 3 and 4 (Figure 4, compare panels B and C).
These backbone positions are immediately adjacent to
the scissile phosphate, and form part of the most
deformed region of the I-PpoI:DNA substrate complex
(21). The selectivity of K61 variants for mutant target
site DNA is likely explained by sequence-dependent
conformation changes in the DNA–protein interface,
as no new contacts are established with mutant target
site DNA.
DISCUSSION
We used a yeast one-hybrid (Y1H) assay to isolate and
characterize variants of the I-PpoI homing endonuclease
with altered DNA target recognition specificity. Variants
were isolated using a mutant binding site target with
symmetrical
6 bp position substitutions that abolished
site binding and cleavage by native I-PpoI. We reasoned
that amino acid substitutions in the DNA–protein inter-
face adjacent to this target site position, at contacting
residue 63 alone or in conjunction with other sub-
stitutions, might restore high site-binding affinity and
Figure 4. Molecular modeling of I-PpoI variant protein on native and mutant target site DNAs. The native
6A/T base pair is shown in yellow,
while the mutant
6C/G base pair is shown in purple. Amino acid geometries pictured with the mutant target site were modeled using
RosettaDesign. Relevant sequence-specific hydrogen bonds are shown as black dotted lines. (A) Geometry of position 6 contacting residue Q63 on
native A:T and mutant C:G target sites. The mutant 6C base (purple) forces Q63 to rotate 1358 to alleviate a stearic clash (shown as crossed arc
lines). (B) Rotation of Q63 forced by the
6C/G base-pair substitution disrupts the canonical R74:guanine contacts made at adjacent base-pair
position 7. (C) Stereo representation of I-PpoI variant G modeled with the
6C/G mutant DNA target. R63 interacts with
5G, and allows R74 to
again contact base pair position 7. R65 makes two H-bonds to target site base-pairs 8 and 9, which increases the overall affinity of I-PpoI for native
and mutant DNA target sites. R61K substitutions alter DNA backbone contacts near the scissile phosphate between base pairs 2 and 3 (compare
panel C with B or Figure 1A).
Nucleic Acids Research, 2007, Vol. 35, No. 17 5847

specificity in vivo. This strategy resembles Y1H screens
that have been used to identify and characterize site-
specific DNA-binding proteins from yeast and other
organisms. One similar precedent reported for homing
endonucleases was the use of a bacterial two-hybrid screen
to explore the recognition specificity of the LAGLIDADG
homing endonuclease PI-SceI (37).
The variant I-PpoI proteins we generated contained
from 1 to 8 amino acid substitutions in the DNA–protein
interface (Tables 1 and 2). Most variants had low site-
binding affinities (100–1000 nM Kd’s), and a modest (2-
to-10-fold) preference to bind native or mutant target
site DNA. One exception, variant T with only a K65R
substitution, had high binding affinities for native and
mutant target site DNAs that included a clear (<175-fold)
preference for native site binding. Cleavage competence
was assayed by reverting the catalytically inactivating
H98A substitution required to establish the Y1H screen,
and then assaying proteins for the ability to cleave native
or mutant target site DNAs. We found little correlation
among site binding, discrimination and cleavage proper-
ties of individual variant proteins (Table 2 and Figure 3,
panels C and D). One likely explanation for this is that I-
PpoI must bind and bend substrate DNA in order to
generate a productive substrate complex (21,22,26). Our
hypothesis is that active variants (e.g. variants U, S or P,
Figure 3A and Table 2) retain the ability to both bind and
bend site DNA to permit cleavage.
Structure-based molecular modeling of I-PpoI variant
proteins on both native and mutant target site DNAs
was used to gain insight into the contribution of specific
residue substitutions to the biochemical behavior of
variant proteins. Modeling revealed that Q63R substitu-
tions fail to confer
6 mutant target site specificity by
virtue of loss of a canonical glutamine:adenine contact at
position 6, together with partial disruption of a canonical
arginine:guanine contact at the adjacent base pair position
7. Q63R variants were nonetheless able to discriminate in
favor of mutant +6C/–6G target sites by packing more
favorably with mutant 6C than with the native 6A target
site base, while making a high quality contact at the
adjacent position 5 base pair (Figure 4). K65R substitu-
tions, in contrast, increase affinity for native and
6
mutant target sites by making an additional H-bond to
bridge base pair positions 8 and 9 [Figures 1 and 4 (panels
B and C)].
R61K substitutions, in contrast, appear to alter site
binding by modifying DNA backbone contacts immedi-
ately adjacent to the scissile phosphate located between
base pairs 2 and 3 [(21); Figure 4]. This type of indirect
readout may be strongly influenced by local, sequence-
dependent DNA conformation (38,39). These sequence-
dependent effects may be further amplified by DNA
substrate deformation in this region of the I-PpoI
substrate complex, and thus interfere with correct position-
ing of the scissile phosphate (Figures 1 and 4) (21,26).
The Y1H screening assay we used employed the
canonical two-hybrid reporters HIS3 and lacZ that,
respectively, confer a growth advantage or have readily
detectable activity at low expression levels. The sensitivity
of these reporters, in retrospect, permitted the
identification of I-PpoI variants with modest in vivo
activity and site-binding affinity (Table 2 and Figure 3).
One explanation for the failure to recover I-PpoI variants
with high affinity and specificity for +6C/–6G mutant
target site DNA is that they were not present in our
starting libraries. This may be the case for the initial
randomized contacting residue (CR) library, which was
too large to be exhaustively screened. The smaller
rationally designed libraries (RD1 and RD2), in contrast,
were exhaustively screened but may have been too small to
encode a high affinity binding variant.
The experimental and computational analyses described
above indicate one productive approach to alter the
DNA recognition specificity of I-PpoI and other homing
endonuclease proteins. Computational DNA–protein
interface design can be used to predict different residue
substitutions that may confer high binding affinity and
specificity for a mutant target site DNA. The resulting
protein variants can then be rank-ordered on the basis of
predicted DNA-binding energies, and further evaluated
for structural quality by molecular modeling. This general
approach has already been shown to work for single base-
pair positions in I-MsoI, a member of the LAGLIDADG
homing endonuclease family (40), and should be
applicable to His-Cys box proteins such as I-PpoI.
A prerequisite for this engineering approach is a high
resolution co-crystal structure.
In addition to protein computational design, it should
also be possible to improve the experimental selection or
screening assays to identify variant proteins with high
site-binding affinity and specificity. For example, the Y1H
assay could be adapted to use reporter genes or selections
that would require comparatively high levels of expression
to identify active variants. Alternatively, a combination of
positive and negative selection could be used to recover
variants with high site-binding affinity and specificity (41).
Homing endonucleases remain the most attractive
starting point for the generation of new, highly
sequence-specific proteins for biology and medicine.
They encode a wide range of different DNA recognition
specificities, and display high site-binding specificity that is
tightly linked to DNA cleavage. Homing endonucleases
including I-PpoI have already been successfully expressed
in human cells. They cleave their target sites with high site
specificity (19,42–44), and thus are being used to promote
site-specific recombination (45) and high resolution DNA
double-strand break repair analyses (20). The precedents
outlined above indicate that it should be possible in the
near future to generate many highly sequence-specific
homing endonuclease variants useful for genome engi-
neering, disease therapy or disease prevention.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Mike Moser, Meggen S. Chadsey and Alden
Hackmann for help, respectively, with experimental
5848 Nucleic Acids Research, 2007, Vol. 35, No. 17

design, library construction and graphics support. Stan
Fields and his laboratory provided generous help in
establishing the yeast one-hybrid assay, and Barry L.
Stoddard for I-PpoI structure determinations, initial
structural modeling and continuous discussion. U.S.
National Institutes of Health RO1 and T32 funding
(RO1 CA88942 to R.J.M., Jr; T32 GM07735 to J.L.E.
T32 GM07270 to J.E. and T32 GM007266 and T32
CA077116 to U. U.). U. U. is also the recipient of a
Poncin Fund Award. Funding to pay the Open Access
publication charges for this article was provided by the
U.S. National Institutes of Health.
Conflict of interest statement. None declared.
REFERENCES
1. Pabo,C.O. and Sauer,R.T. (1992) Transcription factors: structural
families and principles of DNA recognition. Annu. Rev. Biochem.,
61, 1053–1095.
2. Branden,C. and Tooze,J. (1999) Introduction to Protein Structure
Garland Publishing, Inc., New York, USA.
3. Luscombe,N.M., Austin,S.M., Berman,H.M. and Thornton,J.M.
(2000) An overview of the structures of protein-DNA complexes.
Genome Biol., 1, 1–37.
4. Luscombe,N.M., Laskowski,R.A. and Thornton,J.M. (2001) Amino
acid-base interactions: a three-dimensional analysis of protein-DNA
interactions at an atomic level. Nucleic Acids Res., 29, 2860–2874.
5. Sarai,A. and Kono,H. (2005) Protein-DNA recognition patterns
and predictions. Annu. Rev. Biophys. Biomol. Struct., 34, 379–398.
6. Pingoud,A., Fuxreiter,M., Pingoud,V. and Wende,W. (2005) Type
II restriction endonucleases: structure and mechanism. Cell. Mol.
Life Sci., 62, 685–707.
7. Latchman,D.S. (2004) Eukaryotic Transcription Factors Elsevier
Academic Press, London.
8. Belfort,M. and Roberts,R. (1997) Homing endonucleases – keeping
the house in order. Nucleic Acids Res., 25, 3379–3388.
9. Belfort,M., Derbyshire,V., Stoddard,B.L. and Wood,D.W. (2005)
Homing Endonucleases and Inteins Springer, Berlin.
10. Stoddard,B.L. (2005) Homing endonuclease structure and function.
Quarterly Rev. Biophys., 38, 1–47.
11. Porteus,M.H. and Carroll,D. (2005) Gene targeting using zinc finger
nucleases. Nat. Biotechnol., 23, 967–973.
12. Gimble,F.S. (2000) Invasion of a multitude of genetic niches by
mobile endonuclease genes. FEMS Microbiol. Lett., 185, 99–107.
13. Burt,A. and Koufopanou,V. (2004) Homing endonuclease genes:
the rise and fall and rise again of a selfish element. Curr. Opin.
Genet. Dev, 14, 609–615.
14. Muscarella,D.E. and Vogt,V.M. (1989) A mobile Group I intron in
the nuclear rDNA of Physarum polycephalum. Cell, 56, 443–454.
15. Kuhlmann,U.C., Moore,G.R., James,R., Kleanthous,C. and
Hemmings,A.M. (1999) Structural parsimony in endonuclease active
sites: should the number of homing endonuclease families be
redefined? FEBS Lett., 463, 1–2.
16. Galburt,E.A. and Jurica,M.S. (2005) His-Cys box homing
endonucleases. In Belfort,M., Derbyshire,V., Stoddard,B.L. and
Wood,D.W. (eds), Homing Endonucleases and Inteins, Springer-
Verlag, Berlin, Vol. 16, pp. 85–102.
17. Muscarella,D.E., Ellison,E.L., Ruoff,B.M. and Vogt,V.M. (1990)
Characterization of I-Ppo, an intron-encoded endonuclease that
mediates homing of a group I intron in the ribosomal DNA of
Physarum polycephalum. Mol. Cell. Biol., 10, 3386–3396.
18. Ellison,E.L. and Vogt,V.M. (1993) Interaction of the intron-
encoded mobility endonuclease I-PpoI with its target site. Mol. Cell.
Biol., 13, 7531–7539.
19. Monnat,R.J., Hackmann,A.F.M. and Cantrell,M.A. (1999)
Generation of highly site-specific DNA double-strand breaks in
human cells by the homing endonucleases I-PpoI and I-CreI.
Biochem. Biophys. Res. Commun., 255, 88–93.
20. Berkovich,E., Monnat,R.J. and Kastan,M.B. (2007) Roles of
ATM and NBS1 in chromatin structure modulation and DNA
double-strand break repair. Nat. Cell Biol., 9, 683–690.
21. Flick,K.E., Jurica,M.S., Monnat,R.J., Jr and Stoddard,B.L. (1998)
DNA binding and cleavage by the nuclear intron-encoded homing
endonuclease I-PpoI. Nature, 394, 96–101.
22. Galburt,E.A., Chevalier,B., Tang,W., Jurica,M.S., Flick,K.E.,
Monnat,R.J., Jr and Stoddard,B.L. (1999) A novel endonuclease
mechanism directly visualized for I-PpoI. Nat. Struct. Biol., 6,
1096–1099.
23. Flick,K.E., McHugh,D., Heath,J.D., Stephens,K.M., Monnat,R.J.,
Jr and Stoddard,B.L. (1997) Crystallization and preliminary X-ray
studies of I-PpoI: a nuclear, intron-encoded homing endonuclease
from Physarum polycephalum. Protein Sci., 6, 1–4.
24. Argast,G.M., Stephens,K.M., Emond,M.J. and Monnat,R.J. (1998)
I-PpoI and I-CreI homing site sequence degeneracy determined by
random mutagenesis and sequential in vitro enrichment. J. Mol.
Biol., 280, 345–353.
25. Wittmayer,P.K., McKenzie,J.L. and Raines,R.T. (1998) Degenerate
DNA recognition by I-PpoI endonuclease. Gene, 206, 11–21.
26. Galburt,E.A., Chadsey,M.S., Jurica,M.S., Chevalier,B.S., Ehro,D.,
Tang,W., Monnat,R.J, Jr and Stoddard,B.L. (2000) Conformational
changes and cleavage by the homing endonuclease I-PpoI: a critical
role for a leucine residue in the active site. J. Mol. Biol., 300,
877–887.
27. Botstein,D., Falco,S.C., Stewart,S.E., Brennan,M., Scherer,S.,
Stinchcomb,D.T., Struhl,K. and Davis,R.W. (1979) Sterile host
yeasts (SHY): a eukaryotic system of biological containment for
recombinant DNA experiments. Gene, 8, 17–24.
28. Sikorski,R.S. and Hieter,P. (1989) A system of shuttle vectors and
yeast host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics, 122, 19–27.
29. Gietz,R.D. and Woods,R.A. (2006) Yeast transformation by the
LiAc/SS carrier DNA/PEG method. Methods Mol. Biol., 313,
107–120.
30. Sherman,F. (2002) Getting started with yeast. Meth. Enzymol., 350,
3–41.
31. Worthington,M.T., Pelo,J. and Lo,R.Q. (2001) Cloning of random
oligonucleotides to create single-insert plasmid libraries. Anal.
Biochem., 294, 169–175.
32. Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D.,
Seidman,J.G., Smith,J.A. and Struhl,K. (1987) Current Protocols in
Molecular Biology John Wiley & Sons, New York, USA.
33. Miller,J. (1992) A Short Course in Bacterial Genetics: A Laboratory
Manual and Handbook for Escherichia coli and Related Bacteria
Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York, USA.
34. Havranek,J.J., Duarte,C.M. and Baker,D. (2004) A simple physical
model for the prediction and design of protein-DNA interactions.
J. Mol. Biol., 344, 59–70.
35. Morozov,A.V., Havranek,J.J., Baker,D. and Siggia,E.D. (2005)
Protein-DNA binding specificity predictions with structural models.
Nucleic Acids Res., 33, 5781–5798.
36. Muscarella,D.E. and Vogt,V.M. (1993) A mobile Group I intron
from Physarum polycephalum can insert itself and induce point
mutations in the nuclear ribosomal DNA of Saccharomyces
cerevisiae. Mol. Cell. Biol., 13, 1023–1033.
37. Gimble,F.S., Moure,C.M. and Posey,K.L. (2003) Assessing the
plasticity of DNA target site recognition of the PI-SceI homing
endonuclease using a bacterial two-hybrid selection system. J. Mol.
Biol., 334, 993–1008.
38. Lavery,R. (2005) Recognizing DNA. Quarterly Rev. Biophys., 38,
339–344.
39. Koudelka,G.B., Mauro,S.A. and Ciubotaru,M. (2006) Indirect
readout of DNA sequences by proteins: the roles of DNA
sequence-dependent intrinsic and extrinsic forces. Prog. Nucleic Acid
Res. Mol. Biol., 81, 143–177.
40. Ashworth,J., Havranek,J.J., Duarte,C.M., Sussman,D.,
Monnat,R.J., Stoddard,B.L. and Baker,D. (2006) Computational
redesign of endonuclease DNA binding and cleavage specificity.
Nature, 441, 656–659.
41. Doyon,J.B., Pattanayak,V., Meyer,C.B. and Liu,D.R. (2006)
Directed evolution and substrate specificity profile of homing
endonuclease I-SceI. J. Am. Chem. Soc., 128, 2477–2484.
Nucleic Acids Research, 2007, Vol. 35, No. 17 5849

42. Puchta,H., Dujon,B. and Hohn,B. (1993) Homologous recombina-
tion in plant cells is enhanced by in vivo induction of double strand
breaks into DNA by a site-specific endonuclease. Nucleic Acids
Res., 21, 5034–5040.
43. Rouet,P., Smih,F. and Jasin,M. (1994) Introduction of
double-strand breaks into the genome of mouse cells by expression
of a rare-cutting endonuclease. Mol. Cell. Biol., 14, 8096–8106.
44. Lukacsovich,T., Yang,D. and Waldman,A.S. (1994) Repair of
a specific double-strand break generated within a mammalian
chromosome by yeast endonuclease I-SceI. Nucleic Acids Res., 22,
5649–5657.
45. Paˆques,F. and Duchateau,P. (2007) Meganucleases and DNA
double-strand break-induced recombination: perspectives on gene
therapy. Curr. Gene Ther., 7, 49–66.
5850 Nucleic Acids Research, 2007, Vol. 35, No. 17