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 experim
Trang 1In 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
Group I and group II introns frequently contain open
reading frames (ORFs), which are either free-standing
within the intron itself or are in-frame with the preceding 5¢
exon [1,2] While some of these encode essential host
proteins, others have been shown to encode proteins that
facilitate the splicing of their cognate introns These are
called maturases [3]
All known group I maturase proteins are characterized
by two repeated LAGLIDADG amino acid motifs [4]
Interestingly, maturases are highly homologous to a class of
intron-encoded DNA endonucleases (also found in inteins)
[2,5–12] that are characterized by one or two copies of the
same LAGLIDADG motif [7,8,13], reviewed in [7]
Intron-encoded DNA endonucleases catalyze the
mobi-lization of their cognate intron (or intein) into the equivalent
exon sequence of intron-less alleles of the same gene in a
process called homing [14] Significant progress has been
made in the past decade in the characterization of homing
endonuclease biochemistry and several crystal structures
now exist, both with [15–17] and without bound DNA
substrate [18–21]
Homing endonucleases containing one LAGLIDADG
motif (e.g I-CreI) are about half the size of those with two
copies and structural analysis has shown that they function as
homodimers [21] Double motif-containing endonucleases, including the intein-encoded endonucleases SceI and PI-PfuI as well as the archael intron-encoded protein I-DmoI, were crystallized as monomers [19,20] Molecular modeling and crystal structure studies suggest that single-motif, homodimer and double-motif, monomeric LAGLIDADG 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 MgCl2 concentration is >25 mM [29]
We have shown that AnCOB encodes a maturase protein with two LAGLIDADG motifs that specifically and significantly facilitates AnCOB splicing in Mg2+ concen-trations as low as 2 mM [30] Previous genetic evidence indicated that the AnCOB intron is mobile [31] and we have
Correspondence to R B Waring, Department of Biology, Temple
University, 12th & Norris Sts., Philadelphia, PA 19122, USA.
Fax: + 1 215 204 6646, Tel.: + 1 215 204 8877,
E-mail: waring@temple.edu
Abbreviations: ORF, open reading frame; nt, nucleotide.
(Received 6 December 2002, revised 30 January 2003,
accepted 12 February 2003)
Trang 2shown since then that the A nidulans AnCOB-encoded
maturase is also a DNA endonuclease [30] According to
homing endonuclease convention, the protein is called
I-AniI One other in vitro maturase-assisted splicing assay
has been developed recently for the yeast mitochondrial bI3
intron ORF, but in this interesting case, the maturase
requires the assistance of a nuclear-encoded protein and
lacks endonuclease activity [32] To our knowledge, I-AniI is
the only protein with which one can biochemically assay
both DNA endonuclease and RNA maturase activities
in vitro This investigation provides the first step in the study
of the relationship between these two distinct activities
Experimental procedures
Expression of I-AniI
I-AniI was expressed and purified as described previously
[30] Purified I-AniI was stored at)20 C in protein storage
buffer [50 mMTris, pH 8, 100 mMKCl, 1 mMdithiothreitol
and 50% (v/v) glycerol] Unless otherwise noted, all reaction
components are indicated at their final concentrations 1 nM
I-AniI has been defined previously in our laboratory as the
concentration of protein that gives a burst of 1 nM 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)
[a-32P]dCTP (New England Nuclear, Boston, MA, USA)
and unincorporated nucleotides were removed using a P-30
spin column (Biorad, Hercules, CA, USA) after organic
extraction The concentration of DNA was measured
spectrophotometrically The following pairs of
oligonucleo-tides (Integrated DNA Technologies, Coralville, IA, USA)
were annealed together to make recognition site variants
containing 5¢-EcoRI and 3¢-HindIII sticky ends: AnI19R
5¢-AATTCATGAGGAGGTTTCTCTGTAACA-3¢; AnI19H
5¢-AGCTTGTTACAG-AGAAACCTCCTCATG-3¢; AnI17R
5¢-AATTCACGAGGAGGTTTCTCTGTACTA-3¢;
TG-3¢; AnI15R 5¢-AATTCACAGGAGGTTTCTCT-GTC
TA-3¢; AnI15H 5¢-AGCTTAGACAGAGAAACCTCC
TGTG-3¢ Each annealed oligonucleotide pair was
subcloned into the equivalent sites of pIBI24 to generate
LE19, LE17 and LE15, respectively Point mutations were
made by altering the sequence of the pair of AnI19
oligonucleotides as required The plasmid construct,
pCOB-sal, used to transcribe the AnCOB RNA substrate, was
generated previously [29] Either PvuII- or
HindIII-linea-rized pCOBsal run-off RNA transcripts were generated, purified by denaturing PAGE and their concentrations quantitated by liquid scintillation counting as previously described [33] The pre-RNA derived from a PvuII-linearized pCOBsal DNA template was 632 nucleotides (nt) in length and contained 311 nt of intron sequence, 112 nt of 5¢ exon and 209 nt of 3¢ exon sequence HindIII-derived pre-RNAs contained a shorter 3¢ exon, 29 nt in length
Endonuclease cleavage assays The standard endonuclease reaction was performed in TK9 buffer (50 mM Tris, pH 9, 50 mMKCl, 1 mM dithiothrei-tol) Except where indicated, TK8 buffer (50 mM Tris,
pH 8, 50 mM KCl, 1 mM dithiothreitol) was used for experiments containing both DNA and RNA For single-turnover protein in excess cleavage reactions, 10 nMI-AniI were mixed with 1 nMend-labeled pCOBLE in TK9 buffer
at 37C for 2 min Reactions were started with MgCl2at a final concentration of 10 mM Using the Marquardt– Levenberg algorithm and nonlinear regression analysis (PSI-Plot), single-turnover endonuclease data were fit to a single exponential, Fpre¼ Ae–kt+ (1) A) Fprerepresents the fraction of DNA remaining, A is the amplitude, k is the first-order rate constant, kobs, and (1-A) represents the fraction of unreacted DNA All DNA cleavage reactions were quenched in 6· stop buffer (1% SDS, 100 mMEDTA, 0.25% bromophenol blue, 30% glycerol) and reaction products were separated on 1% agarose gels, dried under vacuum at 90C and were visualized by autoradiography RNA splicing assays
Single- and multiple-turnover protein-assisted RNA splicing assays were performed in TK8 buffer containing 5 mM
MgCl2, 0.2 U RNA-Guard (Pharmacia, Piscataway, NJ, USA) and 0.5 mM 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 nM) of I-AniI and
1 nM 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 mMMgCl2at 37C with one compo-nent varied as required Fifty millimolar Mes replaced
50 mM Tris for pH optima experiments performed at
pH < 7
Nitrocellulose filter binding assays
To determine the degree to which DNA and RNA saturate I-AniI, 1 nM I-AniI was bound to varying concentrations of end-labeled pCOBLE or uniformly
Trang 3labeled AnCOB pre-RNA for 5 min at 37C in TK8
buffer containing either 2 mM CaCl2or 5 mM MgCl2for
DNA or RNA substrates, respectively Preliminary
experiments indicated that equilibrium was reached during
that time Samples, in triplicate, were subsequently filtered
through pre-wet nitrocellulose filters and were quantitated
as described previously [33] To estimate the stoichiometry
of RNA and DNA binding, the data were analyzed as
described [34] To determine the dissociation rate constant
of the DNA substrate, 2 nM I-AniI were preincubated at
37C for 10 min in TK8 buffer containing 2 mM CaCl2
(this inhibits DNA cleavage) and 1 nM end-labeled
pCOBLE Reactions were then diluted 20-fold in a similar
buffer containing 28 nM 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 reacconcentra-tion 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 kbcleavage
product Therefore, since the DNA substrate was
end-labeled, endonuclease reaction products were quantified by
multiplying the yield of the 1.912 kbcleavage 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
presented in Fig 5C,D where HindIII-linearized DNA
templates were used [33] Aliquots removed from splicing
reactions were analyzed as previously described [33]
Results
I-AniI recognition site determination
The standard endonuclease substrate, pCOBLE [30],
consists of 162 bp of exon sequence spanning from
)97 b p to +65 b p with respect to the AnCOB intron
insertion site (Fig 1A) When incubated at 37C with
10 nM I-AniI in TK9 buffer containing 10 mM MgCl2,
end-labeled pCOBLE (1 nM) was specifically cleaved into
two reaction products, 1.912 kband 1.025 kbin length
(Fig 1B) There was no difference in reaction rates when
the protein concentration was doubled indicating that the
DNA substrate was saturated with protein (data not
shown) Typically, >95% of the starting material reacted
in 5 min under these conditions, yielding an average
maximum single-turnover rate constant (kobs(max)) of
2.5 min)1 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 recognition sequences and consequently tolerate a wide variety of point mutations However, bifunctional matu-rase/endonuclease proteins such as I-AniI might display a different pattern of tolerance compared to other homing endonucleases if they evolved to accommodate both RNA and DNA in the same binding site, as suggested previously [6,23] Therefore, to address the sequence specificity of I-AniI, several individual point mutations were generated that correspond to sequence found in the homologous VinCOB gene from Venturia inaequalis [35] (Table 1)
To increase the sensitivity toward decreased binding affinity of AnCOB/VinCOB chimeric point mutants, I-AniI sequence specificity was characterized within an LE19 background as that construct is already partially impaired in binding The observed reaction rates for most point mutants were reduced four- to 10-fold, compared to the LE19 reference construct (Table 1) By contrast, the identity of the base pair at position )8 is critical; <2% relative activity was observed when LE19A-8G was evaluated as a DNA substrate The data therefore indicate that I-AniI is typical in its tolerance to mutation in its recognition site
Thein vitro biochemical properties of I-AniI
In this study we evaluated the biochemical properties of the I-AniI endonuclease reaction to first address whether acquiring maturase activity had significantly altered the endonuclease characteristics of I-AniI, compared to other homing endonucleases, and secondly to establish conditions whereby maturase and endonuclease activities could be studied simultaneously
To determine optimal conditions for DNA cleavage by I-AniI, MgCl concentration, pH, temperature and ionic
Trang 4strength 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, Mg2+ is an essential
cofactor for I-AniI endonuclease activity I-AniI activity
was optimal in approximately 12.5 mMMgCl2, b ut when
MgCl2 was omitted from the reaction, no cleavage was
observed (Fig 2A) Two additional group IIa divalent
cations (Mn2+and Ca2+) were evaluated Mn2+
substi-tuted for Mg2+with similar optima trends, although the
absolute activity was lower than with Mg2+ In contrast,
2 mMCa2+did not support cleavage and was completely
inhibitory in 10 mMMgCl [36]
The pH optimum for DNA cleavage activity was approximately 9 (Fig 2B) Interestingly, at the physiologi-cal pH of 7.5 in yeast mitochondria [37], the relative activity was only around 30% No DNA cleavage activity was observed below pH 6
I-AniI exhibited a broad temperature optimum between
45 and 60C (Fig 2C) In the physiological temperature range, roughly 55% activity relative to the maximum was observed I-AniI was completely inactivated after 2 min at
65C [36]
Although not absolutely necessary for catalytic activity, monovalent cations are required for efficient DNA cleavage activity I-AniI activity was optimal in 25 mM 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 ) was incub ated with 1 n 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 ) and 0.2 n M
pCOBLE (h) reacted 24% slower, indicating that protein concentration was subsaturating.
Trang 5(Fig 2D) The relative endonuclease activity was only about
10% when KCl was omitted from the reaction mix Other
monovalent salts were also evaluated (NH4Cl, NaCl) and
similar trends were observed (data not shown) although
lower relative activities were observed compared to KCl
These studies revealed that the buffer used previously to
characterize I-AniI maturase-assisted RNA splicing
[30,33,38] was unsuitable for DNA cleavage by I-AniI
Therefore, TK8 buffer was chosen to simultaneously study maturase and endonuclease activities, apart from experi-ments the results of which are shown in Fig 5A–D Many group I intron-encoded endonucleases are unstable under standard assay conditions but some can be stabilized either by target site DNA [39] or by nonspecific DNA [37,40,41] Moreover, Mg2+has been shown to partially stabilize at least one intron-encoded endonuclease [39] Therefore, we evaluated I-AniI endonuclease stability in TK9 buffer at 37C
When incubated alone in the standard reaction buffer without MgCl2, around 50% of endonuclease activity was lost within approximately 15 min (Fig 3) The addition of
10 mM MgCl2 to the preincubation mix stabilized endo-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)1BSA 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 MgCl2) 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.
Construct Relative activity
Fig 2 Optimization ofI-AniI endonuclease cleavage reaction Four parameters (A) MgCl 2 concentration, (B) pH, (C) temperature and (D) KCl concentration, were evaluated for their effects on pCOBLE cleavage by I-AniI at 37 C under single-turnover conditions with subsaturating protein concentrations Relative activity in each panel was normalized to a reaction showing the greatest product accumulation at a single time point Unless otherwise noted, all experiments contained TK9 buffer with 10 m MgCl and were performed at 37 C.
Trang 6RNA and DNA substrates bind I-AniI with the same
stoichiometry
To determine the relative stoichiometry of RNA and DNA
binding, nitrocellulose filter binding assays were performed
(Fig 4A) Both DNA and RNA substrates saturated
limiting amounts of protein in the same manner with a
stoichiometry close to 1 : 1 Two independent
determina-tions were performed for each nucleic acid substrate and
yielded an average ratio of DNA/protein of 1.09 : 1 and an
average ratio of RNA/protein of 1.12 : 1
Multiple-turnover (substrate in excess) DNA cleavage
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 nMI-AniI When cleavage reactions were
started with MgCl2, 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 nM) gave a ratio of DNA/protein of 0.94 : 1 As will be
discussed further, these data indicate that both RNA and
DNA b ind I-AniI with a ratio of 1 : 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
actually contain more than one nucleic acid binding site
We previously developed a novel system to evaluate the binding of AnCOB RNA mutants to I-AniI by using them
as specific inhibitors of protein-assisted native AnCOB splicing at low Mg2+concentrations [33] In this study, we hypothesized that if both nucleic acid substrates share one binding site, then maturase or endonuclease activity could
be blocked by a specific nucleic acid inhibitor For the experiments discussed below, varying amounts of either AnCOB pre-RNA or linearized pCOBLE DNA were prebound to I-AniI and either DNA cleavage or protein-assisted RNA splicing activity was subsequently evaluated With increasing (subsaturating) concentrations of pre-bound, wild-type, competitor AnCOB pre-RNA, DNA cleavage by I-AniI was stoichiometrically inhibited (Fig 5A), reaching maximal inhibition with a saturating (3 nM) concentration of AnCOB RNA The sensitivity of the assay was such that 0.1 nMresidual activity would have been detected (Fig 5B) Inhibition was not due to nonspe-cific binding of degraded RNA as RNAs from endpoint
Fig 4 DNA and RNA substrates saturate I-AniI with the same stoi-chiometry (A) Nitrocellulose filter binding assay in TK8 buffer with
1 n M 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 MgCl 2 or with 1 n M
end-labeled pCOBLE DNA cleavage reactions were subsequently
started with the addition of the missing reaction component Ordinate
values were calculated by normalizing the amount of product formed
to a control reaction that was not preincubated.
Trang 7aliquots were completely intact when resolved on
denatur-ing polyacrylamide gels Inhibition was also not due to
trapping of the DNA substrate by the RNA, because
increasing the DNA concentration from 1 to 6 nM (with
3 nM RNA) did not lead to any DNA cleavage Tight
stoichiometric binding of RNA to protein is consistent with
a Kd< 10 pMunder similar conditions, but at pH 7.5 [38]
Additional control experiments were performed to
determine the specificity of endonuclease inhibition by
prebound RNA AnCOBDP9 pre-RNA lacks the short
(18 nt) stem loop P9 and acts as a weak competitive
inhibitor of RNA splicing [33], but when 5 n AnCOBDP9
pre-RNA was prebound to 3 nMI-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 mM KCl [33], we re-evaluated AnCOBDP9 under more stringent conditions When the concentration of KCl was increased from 50 mM to
150 mM, 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 mM 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 2 DNA cleavage reactions were subsequently started with 1 n M end-labeled pCOBLE (see Experimental procedures for details) (B) Control experiments to demonstrate that cleavage of 1 n M end-labeled pCOBLE substrate DNA is sensitive to the concentration of I-AniI The ordinate represents the fraction of substrate DNA that remained after a 2.5 min cleavage reaction (C) Control experiments to demonstrate the specificity of endonuclease inhibition by prebound AnCOB RNA The assay of 5A was repeated with and without 5 n M AnCOBDP9 pre-RNA and with KCl at 50, 100 and 150 m M Note that increasing KCl concentration slows the cleavage of the DNA substrate in the absence of RNA (see also Fig 2D) and that the addition of competitor RNA to an endonuclease reaction masked the 1.025 kbDNA cleavage product (see Fig 1B and Experimental procedures for details) (D) DNA cleavage reactions with and without prebound AnCOBDP9 (data derived from panel C) (E) Substrate RNA does not inhibit endo-nuclease activity when the DNA substrate is prebound I-AniI (8 n M ) was incubated with 0.8 n M end-labeled DNA substrate at 37 C in TK8 buffer containing 10 m M MgCl 2 After 0.5 min the reaction was diluted 20-fold (arrow) into a chase buffer containing either 10 n M prelinearized DNA substrate (j) or 12 n 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.
Trang 8binding affinity to AnCOBDP9 and a trace of splicing
activity [33], also only slightly inhibited endonuclease
cleavage in 150 mM 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 mMKCl The second group I intron from the
A nidulanscytochrome oxidase gene (NOX2), which does
not bind I-AniI [30], did not detectably inhibit DNA
cleavage in 50 mMKCl; 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 37C (Fig 5E) for 0.5 min,
during which time 15% (0.12 nM) of the DNA reacted The
reaction was subsequently diluted into a chase buffer
containing a large excess of either unlabeled AnCOB
pre-RNA or unlabeled prelinearized DNA The control
reac-tion, performed with excess DNA, which prevents any
subsequent binding of labeled DNA, showed that a
significant fraction of labeled DNA remains bound long
enough to react This fraction is not detectably reduced by
the presence of excess RNA indicating that the RNA cannot
inhibit the cleavage of the DNA if the DNA is already
bound By contrast, when 10 nM end-labeled DNA and
3 nM AnCOB pre-RNA were simultaneously added to a
limiting concentration of protein (2 nM) in the same buffer
conditions, <1% DNA cleavage was detected (data not
shown) The significance of this will be discussed later
Pre-bound DNA substrate does not inhibit maturase
activity
The data derived from the endonuclease competition
experiments were consistent with the simplest hypothesis
that both nucleic acid substrates compete for the same single
discrete binding site, as the fraction of cleaved substrate
DNA varied inversely with competitor RNA concentration
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 nM) 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 nM, 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 condiconcentra-tions of the competiconcentra-tion 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 react at the ob served rate >1 nM free protein must have become available within <0.5 min (j, Fig 6B)
To estimate the amount of free protein in the above experiment, we determined the rate at which the pCOBLE DNA inhibitor dissociated from the protein using a nitrocellulose filter binding assay For those experiments,
1 nMlabeled DNA substrate was prebound to 2 nMprotein under the reaction conditions of the inhibition experiment (Fig 6) The rate of the release of bound, labeled DNA was then followed after adding excess unlabeled, pCOBLE DNA (Fig 6D) A koffof 0.07 min)1was measured, nearly two orders of magnitude lower than the splicing reaction (Fig 6B) arguing that very little of the RNA reacts with free protein and that the majority reacts with protein bound to DNA The data are therefore consistent with the hypothesis that both nucleic acid substrates do not bind the same discrete binding site, but suggest that I-AniI contains either two discrete, or overlapping binding sites This will be discussed below
Discussion
The biochemical properties of I-AniI are typical
of homing endonucleases 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, Mg2+-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]) Together, the available data indicate that I-AniI acquired the ability to function as an RNA-binding protein without compromising basic homing endonuclease properties The high pH reaction profile (Fig 2B) displayed by many, if not all, of the LAGLIDADG enzymes may arise
Trang 9from the use of bulk solvent rather than specific enzyme side
chains as a direct general base for activation of the
nucleophilic water [13,15] A highly diverse collection of
basic side chains surround a large solvent pocket which, in
turn, surrounds the active site With such a site, activation of
the water nucleophile would be expected to require a higher
pH than in the case of a canonical restriction endonuclease
that directly deprotonates the same group One possible
advantage for homing endonucleases of such a reaction
mechanism would be an enhanced ability to retain activity
during evolution (because relatively few side chains are
absolutely essential for catalysis) [13] The cost to the
enzyme of this evolutionary advantage would be a pH
optimum that is elevated relative to the surrounding cellular environment
RNA and DNA substrates bind I-AniI with the same stoichiometry
Nitrocellulose filter binding assays, performed side by side for RNA and DNA (Fig 4A) as well as burst size measurements from multiple-turnover DNA cleavage experiments (Fig 4B) and stoichiometric inhibition of DNA cleavage by prebound RNA substrate (Fig 5A) all argue that both nucleic acid substrates bind I-AniI with the same stoichiometry As X-ray crystallography studies
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.
Trang 10indicate that a homing endonuclease with two LAGLI
DADG motifs binds a single DNA molecule [17], our data
argue that RNA binds I-AniI with a stoichiometry of 1 : 1
This is consistent with Solem et al (2001) who assayed
I-AniI RNA binding using a nitrocellulose filter binding
assay and measured protein concentration
spectrophoto-metrically at 280 nm [46] Together, these data justify the
multiple-turnover RNA splicing calibration that we made to
each preparation of protein (see Experimental procedures)
to give a 1 : 1 stoichiometric burst of spliced RNA We note
however, that the active splicing complex could theoretically
consist of two RNA and two protein molecules with each
RNA using two separate domains to bind to a different
region on the two protein subunits
Relationship between endonuclease and maturase
activities
It is generally believed that LAGLIDADG proteins were
originally DNA endonucleases [2,5] (see Introduction) The
fundamental question then arises as to how I-AniI acquired
maturase activity and how in general, proteins might
acquire a new function It is apparent that as I-AniI
acquired the property to function as an RNA maturase, it
maintained properties common to other homing
endonuc-leases suggesting that there have been no dramatic structural
changes in the vicinity of the DNA-binding site that
constitutes a significant portion of LAGLIDADG proteins
[16] Even though the protein-assisted splicing reaction rate
is at least 10 times the rate of self-splicing at the optimal
concentration of Mg2+[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 nMwould
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 nM RNA is present, supports this statement and
suggests that the RNA binding site is still available and its
binding affinity is not altered by the presence of DNA in the
DNA binding site
Interestingly, when 3 nM RNA and 10 nM DNA were
added simultaneously to a limiting amount of protein in
TK8 buffer containing 10 mMMgCl2, DNA cleavage was
not detected (data not shown) The rate constant, k , for
the binding of RNA to protein is equal to 3· 109
M )1Æmin)1 under similar conditions [38] to these We measured the rate constant for the binding of DNA to protein kinetically and obtained a value of at least half this in TK8 buffer containing 10 mM MgCl2 [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 konwould 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 splicing, even when in 50- and 400-fold excess over protein and RNA, respectively (Fig 6B) Interpreting the data as being inconsistent with a single binding site model assumes that the 50-fold excess of DNA nearly saturates the protein prior to the addition of pre-RNA It further assumes that either the dissociation of bound DNA is slow or the 400-fold excess (over RNA) free DNA effectively competes with the labeled pre-RNA to prevent it from binding
Previous nitrocellulose filter binding assays gave an apparent Kdfor DNA and protein of 0.1 nMin the same reaction conditions as Fig 6 [36] The control experiment using a chase of excess unlabeled DNA as presented in Fig 5E can be used to estimate the rate of dissociation and shows that labeled DNA dissociates slowly from I-AniI, with a kobsof 0.17 min)1, which is similar to the value of
koff ¼ 0.07 min)1 as determined by nitrocellulose filter binding assays (Fig 6D) Therefore, these two observations would seem sufficient to suggest that the assumptions 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 Kdby 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