In vitro
analysis of the relationship between endonuclease
and maturase activities in the bi-functional group I intron-encoded
protein, I-AniI
William J. Geese, Yong K. Kwon, Xiaoping Wen and Richard B. Waring
Department of Biology, Temple University, Philadelphia, USA
The AnCOB group I intron from Aspergillus nidulans
encodes a homing DNA endonuclease called I-AniI which
also functions as a maturase, assisting in AnCOB intron
RNA splicing. In this investigation we biochemically char-
acterized the endonuclease activity of I-AniI in vitro and
utilized competition assays to probe the relationship between
the RNA- and DNA-binding sites. Despite functioning as an
RNA maturase, I-AniI still retains several characteristic
properties of homing endonucleases including relaxed sub-
strate specificity, DNA cleavage product retention and
instability in the reaction buffer, which suggest that the
protein has not undergone dramatic structural adaptations
to function as an RNA-binding protein. Nitrocellulose filter
binding and kinetic burst assays showed that both nucleic
acids bind I-AniI with the same 1 : 1 stoichiometry. Fur-
thermore, in vitro competition activity assays revealed that
the RNA substrate, when prebound to I-AniI, stoichio-
metrically inhibits DNA cleavage activity, yet in reciprocal
experiments, saturating amounts of prebound DNA sub-
strate fails to inhibit RNA splicing activity. The data suggest
therefore that both nucleic acids do not bind the same single
binding site, rather that I-AniI appears to contain two
binding sites.
Keywords: Aspergillus nidulans; homing endonuclease;
RNA binding protein; DNA sliding; RNA splicing.

homing endonucleases contain one DNA-binding site and
share roughly the same extended overall structure with either
two- or pseudo twofold symmetry [19–21], reviewed in
[8,13].
The phylogenetic distribution of LAGLIDADG homing
endonucleases is widespread [7] but that of maturases is less
well known. Introns from Saccharomyces cerevisiae encode
proteins with either maturase or endonuclease activity, but
not with both activities [2]. However they are clearly closely
related [22–25] and in vivo assays have shown that the
Saccharomyces capensis cobi2 intron-encoded protein is
both an endonuclease and a maturase [26].
There is evidence that endonuclease ORFs, acting as the
minimal agent of mobility, invaded group I introns [27,28].
The parsimonious argument follows that this eventually
conferred mobility upon the host introns and that maturase
activity subsequently evolved from some endonucleases
[2,5] thus facilitating intron transposition to new sites
[9–12,14,29].
The AnCOB group IB intron from the apocyto-
chrome b gene in Aspergillus nidulans self-splices in vitro,
providing that the MgCl
2
concentration is >25 m
M
[29].
We have shown that AnCOB encodes a maturase protein
with two LAGLIDADG motifs that specifically and
significantly facilitates AnCOB splicing in Mg
2+

M
Tris, pH 8, 100 m
M
KCl, 1 m
M
dithiothreitol
and 50% (v/v) glycerol]. Unless otherwise noted, all reaction
components are indicated at their final concentrations. 1 n
M
I-AniI has been defined previously in our laboratory as the
concentration of protein that gives a burst of 1 n
M
RNA
products in an RNA-splicing reaction performed under
multiple-turnover conditions [30]. This definition was insti-
tuted because similar assays resulted in RNA/protein ratios
of 1 : 1 to 2 : 1 when the concentrations of different protein
preparations were determined by the Bradford Assay.
Subsequent precise calibration using multiple-turnover
RNA splicing assays (see below) preserves uniformity
between different protein preparations. Throughout this
work, only the calibrated protein concentration was used.
Preparation of nucleic acid substrates
The standard DNA substrate, pCOBLE, was generated
previously [30]. A preparative amount of pCOBLE plasmid
DNA was purified over a single CsCl centrifugation density
gradient. Ten micrograms of BsaHI-linearized pCOBLE
was end-labeled with 20–50 lCi (800–3000 CiÆmmol
)1
)

contained a shorter 3¢ exon, 29 nt in length.
Endonuclease cleavage assays
The standard endonuclease reaction was performed in TK9
buffer (50 m
M
Tris, pH 9, 50 m
M
KCl, 1 m
M
dithiothrei-
tol). Except where indicated, TK8 buffer (50 m
M
Tris,
pH 8, 50 m
M
KCl, 1 m
M
dithiothreitol) was used for
experiments containing both DNA and RNA. For single-
turnover protein in excess cleavage reactions, 10 n
M
I-AniI
were mixed with 1 n
M
end-labeled pCOBLE in TK9 buffer
at 37 °C for 2 min. Reactions were started with MgCl
2
at a
final concentration of 10 m
M

guanosine as described previously
[30,33]. Note that all RNAs studied in this investigation
were uniformly labeled. Therefore the yield of each RNA
species in splicing reactions was corrected for the number of
uridines present.
Endonuclease optimization experiments
All endonuclease optimization experiments were performed
using a subsaturating concentration (4 n
M
)ofI-AniIand
1n
M
end-labeled pCOBLE. All reactions were quenched
during a time frame in which product formation varied
exponentially with time and no more than 50% of the
starting material reacted to further ensure that each
determination was sensitive to minor changes in reaction
rate. Optimization experiments were performed in TK9
buffer containing 10 m
M
MgCl
2
at 37 °C with one compo-
nent varied as required. Fifty millimolar Mes replaced
50 m
M
Tris for pH optima experiments performed at
pH < 7.
Nitrocellulose filter binding assays
To determine the degree to which DNA and RNA

M
end-labeled
pCOBLE. Reactions were then diluted 20-fold in a similar
buffer containing 28 n
M
linearized, unlabeled pCOBLE
and the release of the labeled DNA was followed over
time using a nitrocellulose filter binding assay as described
above. A control reaction that did not contain a chase
was also performed. Adding both DNAs simultaneously
gave a negligible signal above background.
In vitro
competition assays
RNA splicing and DNA cleavage competition experiments
both involved a prebinding step in which either DNA or
RNA substrates were incubated with I-AniI in a binding
reaction containing either 1.5· or 1.1· the final concentra-
tion of each reaction component, respectively. RNA splicing
and DNA cleavage reactions were subsequently started with
the missing reaction components in a volume sufficient to
dilute all the reaction components to their final concentra-
tions. When an RNA inhibitor was included in an
endonuclease reaction, it masked the 1.025 kb cleavage
product. Therefore, since the DNA substrate was end-
labeled, endonuclease reaction products were quantified by
multiplying the yield of the 1.912 kb cleavage product by
two. To preserve uniformity, control endonuclease experi-
ments, without competitor RNA, were quantified in the
same way. All pre-RNAs were derived from PvuII-linea-
rized DNA templates except for indicated experiments

over several different protein preparations
(Fig. 1B).
Previous dideoxynucleotide sequencing studies mapped
the boundaries of the I-AniI recognition sequence to
approximately 20 bp [30]. However, in those studies the
recognition sequence was located at the end of the DNA
substrate. In this study, we set out to more precisely
determine the minimum sequence cleaved by I-AniI. We
therefore generated three successively shorter DNA sub-
strates in pIBI24 (LE19, LE17 and LE15) that contain 19,
17 and 15 bp of AnCOB exon sequence surrounding the
I-AniI cleavage site (Fig. 1A). To avoid inadvertently
extending the size of the desired recognition sequence the
oligonucleotides were designed to ensure that 7 bp of
sequence flanking the truncated recognition sequence had
minimal similarity to the omitted native sequence.
DNA cleavage reactions were performed under single-
turnover conditions with protein in excess, but limiting
concentrations. Under such conditions, reduction in either
binding or catalytic proficiency should be reflected by a
concomitant decrease in reaction rate. The LE19 construct
supported significant DNA cleavage activity yielding a
corresponding rate constant approximately 24% of that
observed with the standard 162 bp DNA substrate, pCO-
BLE (Fig. 1C, Table 1). Only trace DNA cleavage activity
was observed when the LE17 construct was evaluated as
substrate (Fig. 1C). The LE15 construct showed no detect-
able activity even in the presence of a 30-fold increase in
protein concentration.
In general, homing endonucleases typically have large

whereby maturase and endonuclease activities could be
studied simultaneously.
To determine optimal conditions for DNA cleavage by
I-AniI, MgCl
2
concentration, pH, temperature and ionic
Ó FEBS 2003 Endonuclease and maturase activities of I-AniI (Eur. J. Biochem. 270) 1545
strength were varied systematically and their effects on
pCOBLE cleavage were assessed under single-turnover
conditions with a limiting concentration of protein. As with
all known homing endonucleases, Mg
2+
is an essential
cofactor for I-AniI endonuclease activity. I-AniI activity
was optimal in approximately 12.5 m
M
MgCl
2
,butwhen
MgCl
2
was omitted from the reaction, no cleavage was
observed (Fig. 2A). Two additional group IIa divalent
cations (Mn
2+
and Ca
2+
) were evaluated. Mn
2+
substi-

KCl
Fig. 1. I-AniI recognition site determination. (A) I-AniI recognition site. 30 bp of AnCOB exon sequence flanking the intron insertion site (arrow)
are shown. The cleavage site is indicated with a staggered line. The boundaries of the three truncation mutants, LE19, LE17 and LE15 are indicated
above. Residues that were mutated to the corresponding VinCOB sequence (Table 1) are indicated in lowercase. (B) Typical DNA cleavage reaction
under single-turnover conditions. I-AniI (10 n
M
)wasincubatedwith1n
M
end-labeled pCOBLE in TK9 buffer containing 10 m
M
MgCl
2
at 37 °C.
(C) Single-turnover, subsaturating endonuclease cleavage reactions with varying DNA substrates in TK9 buffer containing 10 m
M
MgCl
2
and 10%
glycerol. Reactions containing 6 n
M
I-AniI and 0.3 n
M
DNA are indicated (d, r, m, j). A control reaction with 33% less I-AniI (4 n
M
)and0.2 n
M
pCOBLE (h) reacted 24% slower, indicating that protein concentration was subsaturating.
1546 W. J. Geese et al.(Eur. J. Biochem. 270) Ó FEBS 2003
(Fig. 2D). The relative endonuclease activity was only about
10% when KCl was omitted from the reaction mix. Other

nuclease activity, with 50% activity lost in about 45 min.
The substitution of either Na
+
for K
+
or acetate for Cl
–
ions, as well as the inclusion of 0.1 mgÆmL
)1
BSA did not
increase the stability of the protein and slowed the reaction
rate (data not shown). The inclusion of 5–10% glycerol
increased stability two- to fivefold, depending on the
preparation of protein, although its inclusion slows the
reaction rate by about 30% (data not shown). Strikingly,
preincubation with linearized pCOBLE DNA substrate
(without MgCl
2
) preserved, upon extrapolation, 50%
activity for approximately 2 h. Pre-incubation with linea-
rized nonspecific (vector) DNA did not detectably stabilize
the protein (data not shown).
Table 1. I-AniI recognition site sequence specificity. Experiments were
performed using end-labeled pCOBLE variants (lowercase letters
in Fig. 1A) as substrates for DNA cleavage reactions under single-
turnover conditions with subsaturating protein concentrations, as
described in the legend to Fig. 1C. The primary data were fit to a single
exponential as described in Experimental procedures. Relative activity
(with respect to LE19) reflects the average of two independent
determinations.

reactions were also performed to estimate the stoichiometry
of DNA binding (Fig. 4B). In those experiments, two
different concentrations of end-labeled pCOBLE were
incubated with 3 n
M
I-AniI. When cleavage reactions were
startedwithMgCl
2
, a small rapid burst was observed. This
was followed by a much slower phase, which is believed to
result from slow release of the cleavage products from the
protein. The amplitudes of the initial burst (2.78 and
2.83 n
M
) gave a ratio of DNA/protein of 0.94 : 1. As will be
discussed further, these data indicate that both RNA and
DNAbindI-AniIwitharatioof1:1.
Pre-bound RNA substrate inhibits endonuclease
activity
It has been hypothesized that bifunctional maturase/endo-
nuclease proteins utilize the same binding site for DNA and
RNA substrate binding [2]. Indeed the RNA helices that
flank the splice sites of group I introns (P1 and P10) are
similar in sequence to part of the endonuclease recognition
sequence [42]. However, chemical mapping studies [38] as
well as RNA mutational analysis [33] indicated that I-AniI
binds multiple RNA domains within the AnCOB intron.
This suggests that the overall tertiary structure of I-AniI may
differ significantly from that of other characterized homing
endonucleases and raises the possibility that I-AniI may

I-AniI and varying concentrations of end-labeled pCOBLE or
uniformly labeled AnCOB pre-RNA. RNA binding reactions con-
tained 5 m
M
MgCl
2
and DNA binding reactions contained 2 m
M
CaCl
2
. Both determinations were performed in triplicate and were
made using the same diluted aliquot of the same protein preparation.
(B) Multiple-turnover endonuclease cleavage assay in TK9 buffer
containing 10 m
M
MgCl
2
. Reactions contained 3 n
M
I-AniI and either
30 n
M
or 60 n
M
end-labeled pCOBLE.
Fig. 3. I-AniI stability in endonuclease reaction buffer. A subsaturating
concentration (3 n
M
) of I-AniI was incubated at 37 °C alone in TK9
buffer with no additional component, with 10 m

M
AnCOBDP9
pre-RNA was prebound to 3 n
M
I-AniI as described above
in TK9 buffer, it significantly inhibited DNA cleavage
activity (Fig. 5C,D). As RNA splicing inhibition studies
were performed in 100 m
M
KCl [33], we re-evaluated
AnCOBDP9 under more stringent conditions. When the
concentration of KCl was increased from 50 m
M
to
150 m
M
, there was only limited inhibition of DNA cleavage
by AnCOBDP9 pre-RNA (compare open symbols in
Fig. 5D). By contrast, there was complete inhibition by
the intact AnCOB pre-RNA in 150 m
M
KCl (data not
shown), indicating that the inhibition was specific. Another
deletion mutant preRNA, AnCOBDP9.1, with similar
Fig. 5. Pre-bound RNA substrate stoichiometrically inhibits DNA cleavage. (A) I-AniI (3 n
M
) was incubated with or without uniformly labeled
AnCOB pre-RNA at the indicated concentrations for 5 min at 37 °C in TK9 buffer containing 10 m
M
MgCl

M
unlabeled pre-RNA substrate (m). A control reaction that was not diluted in a chase buffer, but was left to react to
completion is indicated (d). No end-labeled DNA substrate reacted when the reaction was started under chase conditions.
Ó FEBS 2003 Endonuclease and maturase activities of I-AniI (Eur. J. Biochem. 270) 1549
binding affinity to AnCOBDP9 and a trace of splicing
activity [33], also only slightly inhibited endonuclease
cleavage in 150 m
M
KCl whereas an inactive deletion
mutant AnCOBDP5aiib (described in [33]), which bound
less tightly to I-AniI [33], inhibited DNA cleavage poorly
even in 50 m
M
KCl. The second group I intron from the
A. nidulans cytochrome oxidase gene (NOX2), which does
not bind I-AniI [30], did not detectably inhibit DNA
cleavage in 50 m
M
KCl; nor did a 224 nt RNA transcribed
from the transcription vector (pSP65) (data not shown). In
general, when analyzed at a suitably stringent concentration
of KCl, there was a correlation between inhibition of
endonuclease activity and inhibition of splicing activity
indicating that RNA binding can be monitored by meas-
uring inhibition of the endonuclease reaction [36].
In the above experiments, the RNA was prebound to
I-AniI before addition of the DNA substrate. To determine
whether the RNA substrate can inhibit the cleavage of
prebound DNA substrate, a single-turnover DNA cleavage
reaction was incubated at 37 °C (Fig. 5E) for 0.5 min,

However, in striking contrast to the results described above,
when 50-fold excess (over protein) pCOBLE DNA was
prebound to I-AniI, the rate of protein-assisted splicing of
AnCOB RNA was not significantly different from the rate
of a reaction that did not contain competitor DNA
(Fig. 6A,B). Within the first 0.25 min, around 42%
(0.1 n
M
) of the pre-RNA reacted with or without prebound
DNA. We investigated the robustness of this experiment by
performing it independently a total of three times using two
different preparations of protein and three different
preparations of DNA and RNA substrates. Each time the
splicing reaction was minimally affected by prebinding the
DNA to protein.
As the concentration of protein used, 2 n
M
, yields a rate
of splicing which is about 25% that of the maximal rate [33]
and is therefore subsaturating, any reduction in reaction
rate caused by prebinding the DNA was expected to be
clearly detectable. Control protein-assisted RNA splicing
assays, without competitor DNA, confirmed that the
reaction rate was indeed dependent on protein concentra-
tion under the conditions of the competition assays
(Fig. 6C). A comparison of Fig. 6B,C indicates that if
protein-dependent splicing requires dissociation of bound
DNA, then in order for the reaction with prebound DNA to
reactattheobservedrate>1n
M

The recognition site length requirement as well as sequence
specificity of other homing endonucleases have been deter-
mined [39,43,44]. In this study, the minimum sequence
cleaved by I-AniI (19 bp) is nearly two turns of the double
helix, which is consistent with the X-ray crystal structure for
I-CreI [15,16]. Despite that extended length requirement,
I-AniI tolerates substitution in its recognition sequence
(Table 1). The sample of I-AniI DNA substrate mutations
show a similar trend to those reported elsewhere, with the
majority having a moderate effect and the occasional
mutation (e.g. LE19A-8G) very significantly inhibiting
cleavage. This indicates that I-AniI has not lost the ability
to relax specificity, the hallmark of a homing endonuclease,
in order to function as a maturase. Moreover, the elevated
pH optimum, Mg
2+
-dependence and instability of I-AniI in
the reaction buffer are typical properties of many group I
intron-encoded endonucleases [36,39,40,43]. Furthermore,
multiple-turnover DNA cleavage experiments yielded a rate
constant (0.25 min
)1
) (Fig. 4B) approximately 10-fold
lower than that observed under single-turnover conditions
with a saturating protein concentration, suggesting that
product release is rate-limiting for the cleavage reaction.
This is consistent with observations made with other
homing endonucleases that remain bound to either the 3¢
exon cleavage product (e.g. I-SceI [45]), or remain bound to
both 5¢ and 3¢ exon cleavage products (e.g. I-CreI [40]).

Fig. 6. Pre-bound DNA substrate fails to inhibit RNA splicing. (A) I-AniI (2 n
M
) was incubated at 37 °C with or without 100 n
M
pCOBLE
prelinearized DNA for 5 min in TK8 buffer containing 2 m
M
CaCl
2
, which inhibits endonuclease activity. RNA splicing assays were subsequently
started with the addition of 0.25 n
M
uniformly labeled AnCOB pre-RNA, 5 m
M
MgCl
2
and 0.5 m
M
guanosine (see Experimental procedures). To
confirm that reaction rates were dependent on protein concentration, control protein-assisted RNA splicing assays, without competitor DNA, were
performed with gradually decreasing concentrations of I-AniI. A control reaction with 0.5 n
M
I-AniI is shown. Separate control experiments
demonstrated that self-splicing (in the absence of protein) does not occur in this reaction buffer (data not shown). (B) Protein-assisted RNA splicing
reactions with and without competitor DNA substrate (derived from panel A). (C) Control protein-assisted RNA splicing reactions, without
competitor DNA substrate (derived from panel A). Note that the reaction containing 2 n
M
I-AniI is the same as in panel B. (D) The DNA substrate
dissociates very slowly from I-AniI. Nitrocellulose filter binding assays were performed as described in Experimental procedures. Binding reactions
with and without a DNA chase are indicated as d and s, respectively.

2+
[30] it would seem most likely that
the maturase activity consists primarily of an RNA binding
site but not a catalytic site. Because the 7 bp helix spanning
the 5¢ splice site (P1) shares partial sequence similarity with
the DNA endonuclease recognition site, one might predict
that I-AniI co-opted a pre-existing nucleic acid (DNA)
binding site to function as an RNA binding protein [23].
However, previous RNA deletion analysis argues that the
protein is recognizing considerably more than just a single
short helical region [33]. As will be discussed below, the
available data are consistent with a two binding site model.
To explore the relationship between endonuclease and
maturase activities, we utilized a novel in vitro competition
model system developed in our laboratory [33]. The
competition assays described in this investigation demon-
strated that prebound RNA substrate efficiently inhibited
DNA cleavage activity only when prebound to I-AniI
(Fig. 5A,E). The failure of pre-RNA to interrupt a DNA
cleavage reaction when added shortly after it begins
(Fig. 5E) argues that DNA cleavage can still take place in
the presence of pre-RNA. Control RNA splicing reactions
showed that the RNA at a concentration of 12 n
M
would
have had sufficient time to bind in this experiment (data not
shown). The rate of the splicing reaction in Fig. 6B, in which
only 2 n
M
RNA is present, supports this statement and

[33]. Therefore, given that the
DNAÆprotein complex is more likely to react than dissociate
(Fig. 5E), theoretically the DNA substrate should have
competed effectively with the RNA when added simulta-
neously if both nucleic acids share a single binding site.
However the protocol used to measure k
on
would capture,
in a given period of time, every binding event to any region
of DNA that eventually leads to cleavage if the protein were
to scan the DNA for the recognition site. If DNA cleavage
involves a two-step process such as this, the pre-RNA could
inhibit the former, but not the latter step by binding to a
separate, RNA specific site. It is possible that when RNA
binds (to its own site) immediately after I-AniI has bound
nonspecifically to DNA, it prevents the protein from
scanning to its recognition site, but does not displace or
inhibit cleavage of the DNA if I-AniI has already reached
the recognition site (Fig. 5E). When a saturating amount of
RNA substrate was bound before the DNA substrate,
DNA cleavage was completely inhibited (Fig. 5A). Assu-
ming a two site model, this could be because DNA sliding is
blocked as just discussed or it may suggest that the RNA
sterically interferes with DNA binding or allosterically
inhibits DNA cleavage. Further experiments are warranted
to address those important issues.
The discussion above is based on the assumption that
both substrates can bind simultaneously to I-AniI providing
that the DNA substrate binds first. This is supported by the
fact that prebound DNA substrate failed to inhibit RNA

outlined above are valid and argues that RNA splicing does
not occur as a result of rapid dissociation of the pro-
teinÆDNA complex. Furthermore, the fact that the protein
was saturated with the same amount of either substrate
(Fig. 4) argues against the trivial explanation that only a
fraction of protein was competent to bind DNA, leaving
another fraction free to react with the RNA.
The control experiment with unlabeled DNA chase as
performed in Fig. 5E as well as experiments performed to
determine the apparent K
d
by filter binding were carried out
with protein in excess. This was not the case for the
experiments in Fig. 4A and Fig. 6D. When the protein is in
1552 W. J. Geese et al.(Eur. J. Biochem. 270) Ó FEBS 2003
excess, this can lead to an overestimation of the strength of
binding because protein molecules bound nonspecifically to
DNA adjacent to the recognition sequence can replace a
protein molecule that has dissociated from the recognition
site. These limitations do not seem sufficient to explain the
complete inability of a saturating amount of DNA substrate
to inhibit RNA splicing (Fig. 6B) and therefore it seems
reasonable to make the working hypothesis that the RNA
and DNA binding sites are not the same, rather that they
are separate. This does not exclude the possibility that both
nucleic acid binding sites may share some degree of overlap.
Careful experimentation to test those hypotheses will need
to be performed to determine the binding characteristics of
the nucleic acid substrates, both alone and in the presence of
one another.

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