From the Carl C. Icahn Institute for Gene Therapy and
Molecular Medicine and the ¶ Department of Microbiology, Mount
Sinai School of Medicine, New York, New York 10029
Received for publication, September 23, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Adeno-associated virus type 2 Rep endonuclease
activity is necessary for both viral DNA replication and site-specific
integration of the viral genome into human chromosome 19. The
biochemical activities required for site-specific endonuclease activity
(namely specific DNA binding and transesterification activity) have
been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is
metal-dependent. To identify this metal-binding site, we
have employed a cysteine mutagenesis approach that targets conserved
acidic amino acids. By using this technique, we provide functional
biochemical data supporting a role for glutamate 83 in the coordination
of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved
in the coordination of metal ions, is closely associated with the
active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published
structural studies of the AAV type 5 endonuclease and suggest that
although the two enzymes are not highly conserved with respect to the
AAV family, their active sites are highly conserved.
AAV21 is a human
parvovirus (for review see Ref. 1) that has the unique ability to
integrate its genome site-specifically into a defined locus of human
chromosome 19q13.4, known as AAVS1 (2-9). This event occurs
under conditions that do not favor productive replication
(i.e. absence of helper virus coinfection), thereby establishing a latent infection (10-12). Although intermediate steps
in the integration process remain undefined, its initiation is thought
to parallel events that occur at the viral origin of replication during
a productive infection (8, 13, 14).
The AAV2 minimal origin of DNA replication consists of two motifs, the
RBS and TRS (6, 15, 16). During viral DNA replication, the virally
encoded Rep protein binds to the RBS. This event is followed by site-
and strand-specific endonuclease activity directed at the TRS. Through
this activity, Rep generates a 3'-hydroxyl group to allow for
unidirectional DNA replication of its hairpinned ends (17). The RBS and
TRS motifs are also present within AAVS1, and together with
Rep have been shown to be both necessary and sufficient for targeted
AAV2 integration at this site (6). Current models suggest that Rep
interacts with the AAVS1 motifs in a manner that is similar
to its interaction with the viral origin of DNA replication thereby
targeting the AAV2 genome for integration at this site (8, 13).
The amino-terminal domain of AAV Rep has been shown to contain all of
the residues necessary for site-specific DNA binding and endonuclease
activity (14, 18, 19). This domain includes an active tyrosine residue,
Tyr-156, which is conserved in all parvovirus nonstructural Rep
proteins. This residue is responsible for the covalent attachment of
Rep during transesterification to the nicked DNA strand through a
5'-phosphotyrosyl linkage to a thymidine residue at the TRS (20, 21).
As expected, mutation of this active site tyrosine results in the
abrogation of TRS nicking activity (19, 22). Both Davis et
al. (19) and our laboratory (14) have demonstrated that Rep
variants consisting of the amino-terminal 200 or 208 amino acids,
respectively, retained specific endonuclease activity. Sequence
comparison between related parvoviruses reveals that Asn-208 is within
a region that is not conserved, suggesting that it may serve as a
linker between the endonuclease and highly conserved helicase domains.
Taken together, these observations suggest that the relevant boundary
with respect to endonuclease function lies close to asparagine 208 and
thus defines the endonuclease domain.
A prerequisite to nicking by Rep is that the TRS is single-stranded,
which in vitro is accomplished via ATP- and
Mg2+-dependent Rep helicase activity (23). On a
single-stranded TRS substrate, helicase activity is no longer a
prerequisite for nicking activity. Therefore, endonuclease activity
itself is ATP-independent. However, the presence of a divalent metal
cofactor is absolutely necessary. Our previous data using the
truncation variant Rep78N208 The existence of an amino-terminal metal-binding site for Rep has
recently been confirmed by the crystal structure of the AAV type 5 Rep
endonuclease domain (25). Hickman et al. (25) have
demonstrated that two histidines and a glutamate residue act to
coordinate the divalent metal ion. Interestingly, type 5 and type 2 appear to be among the most disparate serotypes among the AAV family
members (26). However, using a conditional mutagenesis approach
specific for acidic residues, we demonstrate a metal binding role for
the same glutamate residue, Glu-83 in AAV2, and also provide evidence
that indicates that Glu-164 is likely to be in close proximity to the
active site. Taken together, the coordination of the structural data
for AAV5 Rep and the functional data described here serve to provide an
accurate picture of the AAV2 Rep endonuclease domain.
Cloning of Mutant Rep Expression Constructs--
All mutant Rep
proteins were generated using pHisRep68/15b, which contains the AAV2
rep68 gene in a pET15b vector (Novagen). Site-directed mutagenesis for mutants D14C and E83C were generated via
overlapping PCR using NdeI and SacI for cloning
back into pHisRep68. Mutants E6C, D16C, E36C, D42C, E57C, E75C, E96C,
E114C, E125C, E173C, E184C, E201C, E83A, and E164A were generated using the QuikChange Site-directed mutagenesis kit (Stratagene). Double mutants E83C/Y156F, and E83C/K340H were generated by subcloning sequences carrying one mutation into vectors carrying the complementary mutation. In all cases, sequences generated by PCR were confirmed by sequencing.
Protein Isolation--
All recombinant proteins were isolated
via nickel-nitrilotriacetic acid column chromatography as described
previously (14). 50-ml cultures of BL21(DE3)pLysS cells were prepared
and induced as per the manufacturer's instructions (Novagen), and
induced bacterial pellets were frozen at Endonuclease Assay--
Substrates for endonuclease assays were
made by first kinase labeling the TRS-containing oligonucleotide, RML76
(5-CTAGATGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG-3'), followed by annealing to the appropriate complementary strand, either
RML77 (5'-TCGACAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCAT-3') for
the dsTRS substrate or MY** (5'-TCGACAGTGAGCGAGCGAGCGCGC-3') for
the ssTRS substrate. Nicking assays were performed as described previously (14). Briefly, 20-µl reactions containing 25 mM HEPES·KOH (pH 7.5), 1 mM dithiothreitol,
200 ng of bovine serum albumin, 5 pmol of kinase-labeled substrate,
and, where indicated, 1 mM ATP, 5 mM metal
cofactor, and 100 ng of Rep. Reactions were incubated for 45 min at
37 °C and ethanol-precipitated at Electrophoretic Mobility Shift Assay--
Gel shift substrates
were made by annealing RML76 and RML77 (AAVori) or RML102
(5'-CTTTCCTCGTTGGAATCAGAGCGGGAGCTAAACAGGAGG-3') and RML115
(5'-CGGCCTCCTGTTTAGCTCCCGCTCTGATTCCAAC-3') (RS1) followed by Klenow
labeling with [ DNA Helicase Assay--
The assay was performed as described
previously (14). In a total reaction volume of 10 µl, 50 or 100 ng of
protein was incubated for 30 min at 37 °C in the presence of 30 fmol
of Klenow-labeled AAVori. Each protein was adjusted to 100 ng/µl in
the appropriate storage buffer, and a total of 1 µl of protein and/or
protein storage buffer was added to each reaction. Reactions were
terminated by the addition of 3 µl of loading buffer (10 mM Tris·Cl (pH 7.5), 1 mM EDTA, 0.5% SDS,
0.1% each bromphenol blue and xylene cyanol, and 20% glycerol) and
loaded onto an 8% native polyacrylamide gel in 1× TBE. The
electrophoresed gel was then fixed, dried, and visualized as per the EMSA.
Covalent Attachment Assay--
To visualize the covalently
attached PDCs formed as a result of nicking at the TRS, ssTRS
substrates were used in which the TRS-containing strand (RML76) was
labeled at the 3' terminus using terminal dideoxytransferase (New
England Biolabs) and 3'- Identification of Candidate Residues for Metal
Binding--
Cysteine replacement of acidic residues has been used
previously to identify the metal-binding sites of several endonucleases of the "DDE" motif-containing family. These include TnsA, a subunit of the Tn7 transposase (27), the Tn10 transposase (28), and most
recently, the RAG1 endonuclease which facilitates V(D)J recombination of the immunoglobulin locus (29, 30). This method takes advantage of
the different side chain chemistries between acidic amino acids, aspartate and glutamate, and cysteine with respect to their ability to
interact with divalent metal ions Mg2+ and Mn2+
(31). The oxygen of aspartate and glutamate acts as a good ligand for
both metal ions, whereas the sulfur of cysteine is only able to
interact efficiently with Mn2+. Accordingly, a cysteine
substitution of an acidic amino acid that results in a change in the
divalent metal ion requirement for catalysis strongly implicates that
residue in metal binding.
A multiple sequence alignment of the AAV2 Rep endonuclease domain
(amino acids 1-208) (14) with the nonstructural proteins from
different parvoviruses was performed. This analysis resulted in the
identification of several candidate metal-binding residues (ClustalW
alignment, Blosum similarity matrix, open gap penalty = 10, extend
gap penalty = 0.1). A total of 3 aspartate and 13 glutamate
residues were highly conserved (Fig.
1A). Of these, residues Glu-6,
Asp-14, Asp-16, Asp-42, and Glu-83 were of particular interest, because
it was previously shown that alanine substitution at these positions
abrogated nicking activity, but maintained wild type origin binding
ability (32). Although E164A was shown to have wild type nicking
activity, it was determined that this mutant was unable to mediate
integration in a PCR-based assay and was therefore included in this
group. Alanine substitution at residues Glu-36, Glu-49, Glu-57,
Glu-75, Glu-96, Glu-114, Glu-125, Glu-173, Glu-184, and Glu-201 had no
effect on nicking activity (32) and were controls for this study.
All variants used in this study were cloned into bacterial expression
vectors that place a His6 tag at the amino terminus of the
protein. Recombinant proteins were isolated by nickel-nitrilotriacetic acid affinity column chromatography in comparable quantities and at
greater than 95% purity (Fig. 1B). It should be noted that variant E6C required a lower induction temperature (18 °C) in order
for the protein to be recovered in the soluble fraction.
Nicking Activity of Mutant Rep Proteins--
In order to determine
whether or not the cysteine mutants displayed conditional activity in
the presence of either Mg2+ or Mn2+, TRS
endonuclease assays were performed using two related substrates (Fig.
2). Both substrates contain the AAV2
minimal origin of DNA replication and differ in whether or not the TRS
region is single-stranded (ssTRS, Fig. 2A) or
double-stranded (dsTRS, Fig. 2B). Wild type Rep68
protein retains nicking activity in both Mg2+ and
Mn2+ regardless of the state of the TRS; although
consistent with previous studies, the wild type enzyme clearly favors
Mn2+ on the ssTRS substrate (Fig. 2A,
lanes 2-4, top and bottom) (14, 22).
Mn2+ enhancement of nicking is not as pronounced when the
dsTRS is used (Fig. 2B, lanes 2-4,
top and bottom). Cysteine mutants D14C, D16C, and D42C
follow a wild type-like pattern of nicking in that they retain activity
on both substrates in the presence of either divalent metal ion. E83C,
however, clearly displays a conditional phenotype. Although nicking is
barely detectable in Mg2+ (Fig. 2, A and
B, top, lanes 17-19), it is
recoverable to some extent in the presence of Mn2+ on the
dsTRS substrate (Fig. 2B, bottom, lanes 17-19)
and to significant levels on the ssTRS substrate (Fig.
2A, bottom, lanes 17-19). This switch in
divalent metal ion requirement compared with the wild type enzyme
strongly implicates this residue in coordination of the metal cation.
Although not as remarkable as the conditional phenotype observed for
E83C, E164C nicking activity is also suppressed in Mg2+ but
rescued in Mn2+ (Fig. 2, A and
B, top and bottom, lanes
20-22). Nicking was not observed when a divalent metal cation was
omitted from the reaction or when an endonuclease negative variant,
Y156F, was used (data not shown).
The results from the experiment in Fig. 2 are summarized in Table
I. The total nicking activity of each of
the variants was quantified relative to that of wild type Rep68
on each substrate and metal ion. Overall, that the nicking activity of
E83C and E164C is suppressed in Mg2+ and active in
Mn2+ is apparent, although the extent of rescue is more
pronounced on the ssTRS substrate. Mg2+ is unable to
efficiently support nicking by E83C or E164C on the ssTRS (0 and 11%
of wild type, respectively). For both variants, nicking is restored in
the presence of Mn2+ to approximately half of wild type
Rep68 levels (55% for E83C and 66% for E164C). The control variants,
Glu-36, Glu-49, Glu-57, Glu-75, Glu-96, Glu-114, Glu-125, Glu-173,
Glu-184, and Glu-201 were assayed in the same manner as described in
Fig. 2 and, as predicted, possessed a wild type phenotype in that they
were active in both divalent metal ions (data not shown). This supports
the notion that although Asp/Glu to Cys substitutions are not
conservative, they are tolerated by the enzyme. E6C is an exception, in
that it showed no activity in our assays. As noted above, it was not possible to isolate this mutant under the same conditions as the others, suggesting that this mutation had a more global effect on
protein integrity.
Rescue of E83C Nicking Activity on the dsTRS Substrate--
The
decreased ability of E83C nicking activity to be rescued in
Mn2+ on the dsTRS compared with the ssTRS could be the
result of competition between the putative metal-binding site of the
helicase domain and that of the endonuclease domain on the same metal
cofactor pool. This prospect is magnified because it has been
demonstrated that Rep helicase activity is not efficiently supported in
Mn2+ at the same concentrations as in Mg2+ (21,
33). To overcome this possibility, nicking assays using E83C on the
dsTRS substrate were performed such that both Mg2+ and
Mn2+ were titrated into the same reaction (Fig.
3). Although the wild type enzyme was
active over a broad range of total Me2+ concentrations,
nicking by E83C was only observed when Mn2+ was present in
the reaction (Fig. 3, lanes 13-19). Strong nicking activity
by E83C, comparable with the wild type enzyme, was restored at an
Mg2+ to Mn2+ ratio of 1:1 (Fig. 3, lanes
13 and 17).
The lack of nicking activity in Mg2+ by E83C and E164C
could also be attributed to a decreased affinity for the metal due to the substitution. In order to rule out this possibility, experiments were performed in which each divalent metal cofactor was titrated into
the reaction. This approach assumes that a lower affinity for the
divalent cation could be overcome by an increase in its concentration
in the reaction. As Fig. 4 shows, an
increase in the concentration of Mg2+ on either the ssTRS
(A) or dsTRS (B) substrate did not result in
recovery of nicking activity for either variant, in support of our
hypothesis that the activity of the cysteine substitution mutants is
specifically dependent on Mn2+. The origin of the higher
migrating products in the presence of Mg2+ on the dsTRS
substrate (Fig. 4B, lanes 6, 13, and
19) is unknown. However, it is possible that they may be the
result of a conformational change in the DNA substrate that occurs at
high magnesium concentrations that promote nonspecific nicking by Rep.
A control mutant, E83C/Y156F, did not possess any nicking activity,
thus making the possibility of a copurifying, nonspecific nuclease
unlikely (data not shown).
Conditional Phenotype Is Specific to the Cys-Mn2+
Interaction--
In order to verify that the conditional phenotype
displayed by E83C and the partially conditional phenotype of E164C was
specific for the presence of cysteine at these positions, single
alanine substitutions of these residues were made. As expected, mutant E83A nicking activity was shown to be severely impaired (Fig. 5, lanes 12-16). However,
there is residual activity in the presence of Mn2+ (Fig. 5,
lane 14), suggesting that Glu-83 does not act alone in metal
binding. The generation of E164A allowed us to determine the relative
contribution of this residue to nicking when compared with wild type
Rep68 and Glu-83 variants. Because the nicking phenotype of E164C was
not fully conditional, we hypothesized that this residue may not play a
direct role in metal binding but may perhaps act to maintain the
integrity of the metal-binding pocket within the enzyme. This
hypothesis is based on the position of Glu-164 and its relative
proximity to the active tyrosine residue at position 156 with respect
to the amino acid sequence. However, E164A activity was only slightly
affected when compared with the wild type enzyme, indicating that
Glu-164 does not play a significant role, if any, in metal
interactions. Other divalent metal cations were tested for their
ability to support nicking by the conditional mutants.
Zn2+- and Ca2+-supplemented buffers were also
used for the nicking assay, respectively. Wild type Rep showed very
weak activity in Ca2+, in addition to the stronger activity
typically observed in Mg2+ and Mn2+. However,
nicking by the cysteine-substituted enzymes was only efficiently
supported by Mn2+, as expected. Zn2+ was unable
to support nicking by any of the proteins tested, as reported
previously (21, 34-36).
Glu-83 and Glu-164 Do Not Play a Role in Origin DNA Binding or
Helicase Activity--
Because origin binding and helicase-mediated
extrusion of the TRS (on the dsTRS substrate) are prerequisites to
endonuclease activity, it is possible that the conditional phenotype
observed for E83C and the partially conditional phenotype for E164C are not specific to endonuclease activity per se but have their
effect on the steps prior to transesterification at the TRS. To rule out this possibility, E83C and E164C were tested for the origin binding
and helicase activities in separate assays under conditions that did
not favor their nicking activity, i.e. in buffers lacking Mn2+. Electrophoretic mobility shift assays (EMSAs) were
performed using wild type Rep68 and the nicking mutant Rep68Y156F as a
control. This mutation has been shown previously to have no effect on
origin binding or unwinding and is thus consistent with a catalytic
endonuclease mutant (19, 22). Both E83C and E164C retain wild type
origin-binding affinities in the absence of Mn2+,
demonstrating the independence of this activity from Mn2+
(Fig. 6A). The same held true
for helicase activity; both mutants were able to unwind an AAV2 origin
DNA substrate efficiently and independently of Mn2+, using
Mg2+ as the metal cofactor (Fig. 6B). In order
to demonstrate that the helicase activity was Rep-specific, a
helicase-negative variant, Rep68E83C/K340H, was also included. This
variant was purified in the same manner as the cysteine mutants and, as
expected, did not possess any helicase activity (Fig. 6B).
Taken together, these data demonstrate that Glu-83 and Glu-164 do not
play roles in origin binding or unwinding and thus represent catalytic
residues with respect to endonuclease activity.
E83C and E164C Do Not Affect the Ability of Rep to Covalently
Attach to Origin DNA--
We tested the ability of E83C and E164C to
form covalently attached protein-DNA intermediates characteristic of
transesterification directed by the active site tyrosine at the TRS
(Fig. 7). Covalent attachment assays
illustrate that the formation of stable PDCs correlates to the nicking
activity of the mutants in the presence of either Mg2+ or
Mn2+. E83C and E164C are able to produce the PDC in amounts
relative to their ability to nick the origin substrate in either
Mg2+ or Mn2+. For instance, E83C is only able
to form the PDC in the presence of Mn2+. As a control,
Y156F, the active tyrosine mutant, shows no propensity for forming the
PDC, as the linking tyrosine residue is no longer present. These
results demonstrate that the catalytic integrity of nicking mediated by
E83C and E164C is apparently unaffected by cysteine substitution.
Although endonuclease activity mediated by an active tyrosine
residue is typically metal-independent, we found nicking activity by
the Rep amino-terminal domain to require a divalent metal cation (14).
This suggested that the active tyrosine residue did not act alone in
mediating catalysis, and an unidentified metal-binding site existed
within this domain. We set out to identify the residue(s) that
conferred this metal dependence in order to better define the active
site of the endonuclease domain. The amino acid sequence of the protein
did not reveal any homologies with known endonuclease families to aid
us in the prediction of potential metal-binding sites. Thus, we turned
to a mutational analysis technique geared toward elucidating residues
that interact with divalent metal cations.
The cysteine mutagenesis approach is contingent on the ability of the
wild type enzyme to be active in vitro using not only magnesium, but also manganese as the divalent metal cofactor. In our
hands, the endonuclease domain variant Rep68N208 Parvovirus Rep proteins have been associated with the family of
rolling-circle replication initiator proteins. Characteristic of this
class of proteins is the position of the active tyrosine residue as
well as a cluster of histidine residues that are similar to the
metal-binding motif used by several metalloproteinases (37). Mutational
analysis has suggested that this putative "HuH" motif (where
"u" is any hydrophobic amino acid) is indeed critical for Rep
endonuclease activity (32). The definite role of this motif within the
parvovirus non-structural proteins has now been elucidated by the
recent publication (25) of the AAV type 5 endonuclease domain crystal
structure. The structure indicates that the two histidine residues
comprising rolling-circle replication motif 2 act together with
glutamate 82 (Glu-83 in AAV2) to directly coordinate the divalent metal
ion necessary for endonuclease activity. This confirms our prediction
that glutamate 83 in AAV2 coordinates the metal ion.
The AAV5 Rep endonuclease structure also simplified the interpretation
of mutations whose phenotypes were less clear. For instance, the
difficulty in purifying E6C and its subsequent lack of nicking activity
suggested that this residue was critical for maintaining the structural
and/or functional integrity of the protein with respect to endonuclease
activity. Indeed, it is proposed that glutamate 6 in AAV5 Rep is
necessary for the proper orientation and reactivity of one of the
active site histidine residues. Also noteworthy was the apparently
unaffected phenotype of AAV2 Rep variants D14C, D16C, and D42C, because
alanine substitutions at these positions have been shown to completely
abrogate nicking activity (32). Examination of the three-dimensional
AAV5 Rep structure revealed that these residues are positioned on the
outer surface of the endonuclease domain in relatively unstructured regions. Asp-42 has been implicated previously in Rep oligomerization (38), and it remains possible that the endonuclease domain acts as a
multimer, as has been suggested (23). This notion is supported by the
fact that elimination of a charged residue in this position (i.e. alanine substitution) could result in the loss of
oligomerization. Although no evidence has directly implicated Asp-14
and Asp-16 in such a role, these residues may also contribute to
protein-protein interactions, based on their positions within the
endonuclease structure. Alternatively, alanine substitutions at these
positions may disrupt the overall folding of the protein leading to the observed loss in activity, whereas cysteine substitution did not.
Although our data clearly supported a direct role for Glu-83 in metal
binding, interpretation of the Glu-164 data was less straightforward.
It appeared counterintuitive that cysteine substitution of Glu-164
should alter the divalent metal cation preference of Rep, whereas
alanine substitution barely affected nicking at all. Based on these
data alone, we could not rule out the possibility that Glu-164 may have
a function within the active site. Not surprisingly, the AAV5 crystal
structure reveals that Glu-161 (equivalent to Glu-164 in AAV2) is in
very close proximity to catalytic residues of the enzyme (<5 Å). It
is possible that cysteine in this position slightly disrupts the local
environment of the metal-binding site so that it favors interaction
with Mn2+ over Mg2+. We note that in AAV5 Rep,
Glu-161, Lys-157, and the active site tyrosine at 153 lie within the
same plane. Although a catalytic role for Lys-157 (Lys-160 in AAV2) has
not yet been demonstrated, Hickman et al. (25) suggest that
this lysine residue may be able to interact with the DNA substrate
based on the three-dimensional structure of the active site cleft.
Extending this to Glu-164, especially in light of our data as well as
Ozawa and co-workers (32) implicating this residue in integration,
there may indeed be a catalytic role for Glu-164 in the active site.
These residues together with the active site tyrosine may serve to
mediate downstream steps of the integration process that are not yet
defined and perhaps not addressed by the current standard nicking
assay. Development of a biochemically relevant assay for AAV
integration as well as the resolution of the endonuclease domain
structure complexed with DNA will aid in our understanding of these
residues in the ability of Rep to mediate this complex reaction.
It is to be expected that the active sites of two enzymes from the same
family would be highly conserved. The catalytically relevant elements
of the HuH motif, Glu-6 and Glu-83, and the active site tyrosine are
invariably conserved throughout the endonuclease domains of the
parvovirus non-structural protein family. It is noteworthy that AAV5 is
perhaps the most distantly related of the known AAV serotypes when
compared with AAV2 (26). This disparity is reflected in the Rep
proteins themselves. Although the Rep open reading frames of AAV
serotypes 1, 3, and 4 are over 90% similar to AAV2 Rep, the AAV5 Rep
is only 67% homologous to its AAV2 counterpart. Furthermore, the
endonuclease domain of AAV5 Rep is only 62% similar to AAV2 Rep at the
amino acid level, consistent with the fact that AAV5 Rep cleaves a
different TRS sequence that is not recognized by AAV2 Rep (39). This
suggests that small variations within the structure of the respective
active sites are likely responsible for the differences in target site
specificity between the AAV5 Rep and AAV2 Rep endonucleases. The
crystal structure of the AAV2 Rep endonuclease domain will prove useful
in elucidating the structural determinants of target site specificity
within the respective active sites.
As reviewed by Galburt and Stoddard (40), there are several
possibilities as to the role of a divalent metal cofactor in the
context of endonuclease activity. However, these possibilities are
limited by the fact that direct transesterification by Rep must be
mediated by the active tyrosine residue at position 156. Given this
constraint, a likely role for the divalent metal ion within the active
site may be to activate the hydroxyl group of the active tyrosine for
nucleophilic attack at the TRS or to stabilize the transition state
intermediate of the reaction. Or, as demonstrated previously (41, 42)
for other endonucleases, the metal ion may play a role in target site
selection, perhaps by establishing the proper orientation of target
site sequences within the active site. A role for metal ions in target
site selection is an interesting possibility, as we observed a marked
difference between Mg2+- and Mn2+-mediated
nicking with respect to specificity. Another alternative to metal ions
playing a direct role in transesterification has been proposed for the
Escherichia coli topoisomerase I enzyme. Experiments
performed by Zhu et al. (43, 44) suggest that E. coli topoisomerase I uses a non-DDE-type acidic triad to
coordinate Mg2+ ions. Whereas divalent metal cations are
not necessary for DNA cleavage, Mg2+ interactions at the
acidic binding site elicit conformational changes in the enzyme that
are a prerequisite to relaxing supercoiled DNA. The possibility that
Rep may undergo conformational changes may serve as an additional
explanation for the differences in activity by the Rep variants on the
dsTRS versus the ssTRS substrate. A cysteine-substituted
protein may be unable to convert between different conformational
states efficiently in the presence of Mn2+, i.e.
from helicase to endonuclease, whose activities are required on the
biochemically more demanding substrate (dsTRS).
Further studies are needed to fully understand the role of Rep during
site-specific integration. Of the conserved acidic residues targeted in
this study, our data strongly support a role for Glu-83 in directly
interacting with the divalent cations required for endonuclease
activity. Furthermore, we provide evidence indicating that Glu-164 is
closely associated with the active site. We have shown that these
residues do not play a significant role, if any, in the biochemical
activities that are prerequisites to transesterification directed at
the TRS, namely origin binding and helicase activity, and are therefore
directly related to endonuclease activity. Taken together, it seems
likely that Glu-83 and Glu-164, together with the active tyrosine
residue at position 156, are located within the active site of the AAV2
Rep endonuclease domain, thereby corroborating the data provided by the
AAV5 Rep endonuclease crystal structure. Elucidation of the
amino-terminal metal-binding motif in AAV2 Rep, i.e. the
endonuclease active site, serves as a first step toward understanding
the role of Rep during the integration process and provides a
foundation for further exploring the biochemical role of Rep functions
in mediating viral latency.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
demonstrated that the amino-terminal
domain is able to nick the TRS in a metal-dependent manner.
These observations support that, in addition to the putative
metal-binding site for helicase activity and the carboxyl-terminal zinc
fingers (24), there exists an independent metal-binding site dedicated
to endonuclease activity within the amino-terminal domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C until processed.
Column chromatography, using 1-ml Hi-Trap Chelating columns (Amersham Biosciences), was performed as per the manufacturer's instructions using the His·Bind Buffer kit (Novagen). Proteins were eluted in 300 mM imidazole and desalted over PD-10 columns (Amersham Biosciences) into a buffer containing 25 mM Tris·Cl (pH
7.5), 200 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 20% glycerol.
Proteins were aliquoted and stored at
80 °C until use. Protein
concentrations were estimated by Coomassie Blue staining after SDS-PAGE
using wild type Rep68, quantified by Bio-Rad Protein Assay reagent, as
a reference.
20 °C in the presence of 2 µg of sonicated salmon sperm DNA (Stratagene). Precipitated DNA was
resuspended in 5 µl of formamide loading buffer and fractionated on
15% polyacrylamide, 50% urea gels in 1× Tris·borate·EDTA (pH 8.3).
-32P]dCTP (Amersham Biosciences).
Assays were performed as follows. In a total reaction volume of 14 µl, 100 ng of protein was incubated with 50 fmol of radiolabeled DNA
in a buffer consisting of 0.25× TBE (22.5 mM
Tris·borate, pH 8.3; 0.5 mM EDTA), 1.25 mM
dithiothreitol, 0.05% Nonidet P-40, 500 ng of poly(dI-dC), and 3 µl
of loading buffer (40% sucrose, 1% xylene cyanol, 1% bromphenol blue
in 0.25× TBE). Reactions were loaded onto a 6% native polyacrylamide
gel in 0.25× TBE, incubated an additional 10 min, and run at 18 V/cm at room temperature. Electrophoresed gels were then fixed in 10% trichloroacetic acid, dried, and visualized using a STORM860
PhosphorImager (Amersham Biosciences). Quantification of PhosphorImager
data was performed using ImageQuant version 1.11 software (Amersham Biosciences).
-[32P]ddATP. This
resulted in the addition of a single labeled nucleotide. Nicking assays
were performed as usual, except that the final reaction volume was
adjusted to 10 µl. Reactions were incubated at 37 °C and
terminated by the addition of 1 µl of 0.5 M EDTA and 3 µl of loading buffer. Reactions were run on 6% native polyacrylamide gels in 1× TBE and treated as per the EMSA and helicase assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, conserved acidic residues within the
endonuclease domain of AAV2 Rep targeted in this study. A multiple
sequence alignment of parvovirus Rep proteins revealed several
conserved acidic residues within the amino terminus of Rep. Residues
shown in boldface italics have been implicated in
endonuclease activity previously by charge-to-alanine mutagenesis (32).
GPV, goose parvovirus; MDPV, muscovy duck
parvovirus; SPV, simian parvovirus; ChPV,
chipmunk parvovirus. B, purity of the mutant proteins used
in this study. 8% SDS-PAGE gel with ~1 µg each of the
cysteine-substituted variants. The approximate sizes of the molecular
weight standard (left and center) are indicated
in kilodaltons.
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Fig. 2.
E83C and E164C display a conditional nicking
phenotype. Endonuclease assays were performed on linear AAV origin
DNA substrates that contained either a single-stranded TRS
(A, ssTRS) or a substrate in which the TRS was
double-stranded (B, dsTRS). Reactions were
supplemented with either 5 mM MgCl2
(top) or 5 mM MnCl2
(bottom). Products of the nicking reaction were resolved on
a 15% polyacrylamide, 50% urea (w/v) gel. Under each condition,
lane 1 represents the substrate incubated in the presence of
the indicated metal ion, but in the absence of enzyme; S,
substrate.
Substrate nicking by cysteine mutants in the presence of Mg2+
or Mn2+ divalent metal cofactor.
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Fig. 3.
Optimal rescue of nicking by E83C on the
dsTRS substrate requires both Mg2+ and
Mn2+. Magnesium and manganese ion concentrations were
counter-titrated against each other in standard nicking assays. In each
case, the concentration of one ion was fixed at 5 mM, and
the other ion was titrated in at 5, 10, and 15 mM,
respectively (lanes 4-6, 8-10,
13-15, and 17-29). Lanes 2 and
11 contain reactions incubated without divalent metal
cation, whereas lanes 3 and 12 represent reaction
in which only one ion was present. Lane 1 represents
kinase-labeled marker oligonucleotides corresponding to the expected
products of the nicking reaction incubated under the same buffer
conditions. S, substrate; P, product.
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Fig. 4.
The conditional phenotype of E83C and E164C
cannot be overcome by increasing Mg2+ concentrations.
Nicking activity of wild type Rep68, E83C, and E164C in the presence of
increasing amounts of either MgCl2 (Mg) or
MnCl2 (Mn) as indicated, on the ssTRS
(A) or dsTRS (B) substrate. Lanes 3 and 7 contain 3.125 mM of the indicated metal
cofactor; lanes 4, 8, 12,
14, 18, and 20 contain 6.25 mM
metal cofactor; lanes 5, 9, 13,
15, 19, and 21 contain 12.5 mM
metal cofactor; lanes 6, 10, 16, and
22 contain 25 mM metal cofactor. Lane
1 does not contain enzyme or metal cofactor. Lanes
2, 11, and 17 confirm that nicking is not
observed in the absence of a metal cofactor. S, substrate;
P, product of nicking at the TRS.
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Fig. 5.
Rescue of E83C and E164C Mg2+
suppression is specific for the Cys-Mn2+ interaction.
Nicking assay performed on the ssTRS substrate using alanine- or
cysteine-substituted variants. Lane 1 does not contain
enzyme or metal cofactor. Lanes 2, 12,
17, and 22 confirm that nicking by these mutants is not
observed in the absence of a metal cofactor. S, substrate;
P, product of nicking at the TRS; Zn,
ZnCl2; Ca, CaCl2.
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Fig. 6.
E83C and E164C do not affect origin DNA
binding or unwinding. A, EMSA performed with wild type
or mutant Rep proteins in the presence or absence of specific
(AAV) or nonspecific (RS1) cold competitor DNA at
32-fold excess. Products of the reaction were run on 6% native
polyacrylamide gels. Percent of input substrate shifted in each
reaction is indicated below each lane. Y156F is a
previously described point mutant that is null for nicking activity.
B, helicase assay performed with either 50 or 100 ng of the
indicated protein on the dsTRS substrate. E83C/K340H is the E83C
variant carrying a mutation at lysine 340 that abrogates helicase
activity in the context of the wild type enzyme as a control. Products
of the reaction were run on 8% native polyacrylamide gels. Percent
input substrate unwound in each reaction is indicated below
each lane. S, substrate; P, product of unwinding,
, no enzyme, 100 °C, heat-denatured prior to loading on to the
gel.
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Fig. 7.
The endonuclease activity of E83C and E164C
is able to form covalently attached protein-DNA intermediates.
Covalent attachment assays demonstrating the integrity of the
endonuclease domain of E83C and E164C mutant proteins compared with
wild type Rep68 in the presence of either magnesium (Mg) or
manganese (Mn) as cofactor. Products of the reaction were
resolved on 6% native polyacrylamide gels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was only appreciably active in the presence of Mn2+ ions, and not
Mg2+ (14). For this reason, the full-length Rep68 protein
was used in this study, because it is able to use either
Mg2+ or Mn2+ for nicking activity. In a
multifunctional protein such as Rep, it must be taken into
consideration that a chemically extreme substitution could affect the
integrity of the protein. This concern was especially relevant in this
study, because only evolutionarily conserved residues were targeted.
All but one of the variants tested were apparently unaffected in their
overall structure by the substitution when tested for endonuclease
activity on the dsTRS substrate, which requires both the DNA binding
and helicase activity to be intact.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Peter Ward and Thomas Weber for helpful discussions during the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM/AI62234 (to R. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Training Grants T32AI07647 and T32HD07105.
To whom correspondence should be addressed. Tel.:
212-659-8278; Fax: 212-849-2437; E-mail:
michael.linden@mssm.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M209750200
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ABBREVIATIONS |
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The abbreviations used are: AAV, adeno-associated virus; RBS, Rep-binding site; TRS, terminal resolution site; ss, single-stranded; ds, double-stranded; Me2+, divalent metal cation; PDC, protein-DNA complex; EMSAs, electrophoretic mobility shift assays.
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