Previous Article | Next Article 
Journal of Virology, March 2000, p. 2936-2942, Vol. 74, No. 6
0022-538X/00/$04.00+0
Mutational Analysis of Adeno-Associated Virus Type
2 Rep68 Protein Endonuclease Activity on Partially
Single-Stranded Substrates
Michael D.
Davis,
Jianwen
Wu,
and
Roland A.
Owens*
Laboratory of Molecular and Cellular Biology,
National Institute of Diabetes and Digestive and Kidney Diseases,
Bethesda, Maryland 20892
Received 12 October 1999/Accepted 15 December 1999
 |
ABSTRACT |
The endonuclease activity of the Rep68 and Rep78 proteins
(Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed
by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This
endonucleolytic cleavage is believed to involve the covalent attachment
of Rep68/78 to the trs via a phosphate-tyrosine linkage. A
previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722-2730, 1997) suggested that tyrosine
152 was part of the active site. We individually mutated each tyrosine
within the first 200 amino acids of the Rep68 moiety of a maltose
binding protein-Rep68/78 fusion protein to phenylalanine. Only
mutation of tyrosine 156 resulted in a protein incapable of covalent
attachment to a partially single-stranded hairpin substrate, suggesting
that tyrosine 156 is part of the endonuclease active site.
| |
TEXT |
Adeno-associated virus type 2 (AAV)
is a human parvovirus with a single-stranded, linear DNA genome
containing inverted terminal repeats (ITRs), which function as origins
of replication (11, 32, 33, 41). AAV is nonpathogenic and
usually requires an adenovirus or herpesvirus as a helper for efficient
replication (3). The AAV rep gene encodes at
least four overlapping, multifunctional, nonstructural proteins encoded
by RNA transcribed from two promoters. Rep68 and Rep78 are encoded by
spliced and unspliced transcripts, respectively, from the promoter at
map position 5, hence the first 529 amino acids of Rep78 and Rep68 are
identical (6, 26, 40, 44). Rep40 and Rep52 are encoded by
spliced and unspliced transcripts, respectively, from the promoter at
map position 19 (5).
The AAV ITRs are palindromic and fold into hairpin structures (see Fig.
1) which serve as primers for the synthesis of the complementary strand
(4, 40). The resulting closed-end intermediates are resolved
by a process called terminal resolution, which involves a
site-specific, strand-specific endonuclease cut at the terminal resolution site (trs), followed by unwinding and replication
of the hairpin (13, 37, 39). Rep78 and Rep68 (Rep68/78)
display activities which are required for AAV DNA replication including the abilities to bind specifically to Rep recognition sequences (RRSs)
within the AAV terminal hairpin DNA (13-15, 25, 28, 52) and
to mediate nucleoside triphosphate-dependent, strand-specific nicking
at the trs (13, 15, 37). Rep proteins also have nucleoside triphosphate-dependent DNA helicase (13, 15, 22) and DNA-RNA helicase (53) activities, as well as ATPase
activity (53).
There is strong evidence that Rep68/78 are involved in the preferential
integration of AAV genomes into a region on the q arm of human
chromosome 19 (1, 10, 18-20, 23, 34, 46, 52). This is the
only reported example of site-specific integration in a mammalian virus
system. The chromosome 19 preferred integration locus, designated
AAVS1, contains both an RRS and a trs-like
sequence which can be cleaved by Rep proteins (18, 46, 52).
Linden et al. (23) showed that a 33-bp segment of
AAVS1, containing both the RRS and trs-like
sequence, is sufficient to target AAV integration into an episome. If
either the RRS or trs-like sequence was mutated, then
targeting was lost (23).
Three lines of evidence suggest that the trs endonuclease
activity of Rep68/78 requires a Rep68/78 DNA unwinding activity. First,
Snyder et al. (38) demonstrated that a nucleoside
triphosphate cofactor is no longer required for Rep68 trs
endonuclease activity if the region of the trs is single
stranded. Second, although we have been able to generate several
helicase-positive, endonuclease-negative Rep mutants, we have not been
able to generate any helicase-negative, endonuclease-positive (on a
fully double-stranded hairpin substrate) mutants (9, 22, 28, 48,
49). Recently, Zhou et al. (56) showed that Rep68 can
unwind a blunt-ended, double-stranded DNA substrate if it contains an
RRS. This apparent linkage between the helicase and endonuclease
activities of Rep68/78 has complicated the interpretation of mutational
analyses intended to identify specific amino acid residues involved in
Rep68/78 endonuclease activity (9, 24, 48, 51). By using AAV
hairpin substrates in which the trs is single stranded, we
have uncoupled DNA cleavage from DNA unwinding.
We have used maltose binding protein (MBP)-Rep68/78 fusion proteins
produced in Escherichia coli for this analysis. Our
"wild-type" protein, MBP-Rep68
, contains Rep68/78 amino acids 3 through 522, nearly all of the region which is identical between Rep68
and Rep78 (7). MBP-Rep68 has been shown to have all of
the in vitro functions of Rep68/78 produced in human cells. It binds specifically to DNA containing RRSs (7-9, 48, 49, 54), has
trs endonuclease (7, 9, 46, 48, 49), helicase (7, 9, 48, 49, 53), and ATPase (53) activities, and can complement Rep-deficient cell extracts in an in vitro AAV
replication system (50). Given the ease with which mutant proteins can be generated and purified, we felt that this system was
appropriate for identifying sequences important for the endonuclease activity of Rep proteins.
Protein expression.
MBP-Rep68 fusion proteins were produced in
E. coli containing plasmids encoding these fusion proteins
and purified as described previously (7, 53). Protein
concentrations were determined by optical density at 225 nm using
bovine serum albumin (BSA) standards. The production of MBP-Rep68
proteins of the predicted sizes was confirmed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) and Coomassie blue
staining (data not shown). All of our mutant MBP-Rep68 proteins were
isolated at concentrations and purity levels comparable to the
wild-type protein.
trs endonuclease assays.
The site-specific and
strand-specific endonuclease assay was performed as described
previously (13), with the modifications indicated below.
Plasmid psub201 (31) was used for preparing AAV hairpin DNA
in the flop configuration. psub201 contains a modified AAV genome in
which each ITR is flanked by an XbaI and a PvuII
site. The plasmid was digested with XbaI, followed by dephosphorylation with calf intestine alkaline phosphatase and 32P 5'-end radiolabeling using T4 polynucleotide kinase.
The resulting products were then digested with PvuII,
denatured by heating to 100°C for 6 min, and immediately cooled on
ice for 4 min to form radiolabeled AAV unfilled hairpin in which the
trs is single stranded (Fig.
1). To make filled hairpin, the unfilled
hairpin was treated with Klenow fragment in the presence of dNTPs to
fill in the 5' overhang. The hairpin DNAs were then purified by
nondenaturing PAGE (6% polyacrylamide). For the trs
endonuclease assays, 32P 5'-end-labeled, filled or
unfilled, AAV hairpin DNA (25,000 cpm) was incubated in the presence of
MBP-Rep68 or mutant proteins in a 30-µl reaction volume containing
25 mM HEPES/KOH (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol,
0.3 µg BSA, and 0.4 mM ATP. The reaction mixtures were incubated for
1 h at 37°C and terminated by addition of 15 µl of gel loading
buffer (0.5% SDS, 50 mM EDTA, 40% [vol/vol] glycerol, 0.1%
[wt/vol] bromophenol blue, 0.1% [wt/vol] xylene cyanol) with
subsequent boiling to release the nicked fragment. The reaction
products were resolved on a nondenaturing 6% polyacrylamide gel, which
was then dried and autoradiographed.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 1.
AAV ITR hairpin DNA ("flop" configuration). The
positions of the primary Rep68/78 recognition sequence (RRS)
(52) and the secondary binding site for Rep68/78 (RRS')
(30, 55) are within the labeled rectangles. The individual
imperfect GAGC repeats of the RRS are indicated by subdivisions of its
rectangle. The positions of the terminal resolution site
(trs) (13) and the 3' ends of unfilled hairpin
DNA and partially filled hairpin DNA are also indicated. Fifty-four
base pairs at the right end of the hairpin sequence are indicated by
dots.
|
|
We first wished to test the hypothesis that many of our MBP-Rep68
proteins, containing mutations previously reported to eliminate
trs endonuclease activity on a filled hairpin substrate,
could
nick an unfilled hairpin substrate in which the
trs is
single
stranded. Plasmids encoding MBP-Rep68
, the
D40A-D42A-D44A, E83A-K84A-E86A,
Y121F, K146A-D149A-E150A,
Y152F, G334A, G339A, K340H, T341A, E378A-E379A-K381A,
D402A-K404A-K406A, V418S, D443A-K447A, D455A-D457A, and
K463A-E465A-K467A-D468A
mutant MBP-Rep proteins and MBP-LacZ have been
reported previously
(
7,
9,
48,
49,
53). The designations for
proteins
with amino acid substitutions are the single-letter code for
the
wild-type amino acid, followed by the Rep68/78 amino acid number,
followed by the single-letter code for the new amino acid. The
plasmid
encoding the mutated MBP-Rep68
protein with a deletion
of Rep68/78
methionine 225 (M225
dl) was made by replacing the
PstI-
BamHI fragment of the
rep gene of
pMBP-Rep68
with the corresponding
fragment from
pHIV-Rep/M225
dl (
28).
Figure
2 shows that the Y121F,
M225
dl, G334A, G339A, K340H, T341A, E378A-E379A-K381A,
D402A-K404A-K406A, V418S, D443A-K447A,
D455A-D457A, and
K463A-E465A-K467A-D468A mutant proteins all had
levels of
trs endonuclease activity similar to the wild-type fusion
protein on the unfilled hairpin. The Y152F protein had low but
detectable endonuclease activity. The D40A-D42A-D44A, E83A-K84A-E86A,
and K146A-D149A-E150A proteins had no detectable
trs
endonuclease
activity. An MBP-LacZ fusion protein (
53) also
showed no activity.
View larger version (61K):
[in this window]
[in a new window]
|
FIG. 2.
trs endonuclease assays of wild-type and
mutant MBP-Rep68 fusion proteins on the unfilled hairpin substrate.
trs endonuclease assays were performed (as detailed in the
text, except that no ATP was added) with the mutant fusion proteins
indicated above each lane. Each sample contained 25,000 cpm of 5'
32P-end-labeled unfilled AAV terminal repeat hairpin DNA.
Samples contained either no protein, 1.0 µg of MBP-LacZ (LacZ), or
1.0 µg of the indicated mutant protein. The wild-type (W-T) lanes
contained 1.0 or 0.5 µg of MBP-Rep68. All reaction mixtures were
incubated at 37°C for 60 min, boiled, and resolved on a nondenaturing
6% polyacrylamide gel. The position of the released cleavage product
is indicated on the right.
|
|
It is important to note that only proteins with alterations in the
first 200 amino acid residues of the Rep68/78 moiety were
devoid of
endonuclease activity on the unfilled hairpin substrate.
Care was taken
in the design of the D40A-D42A-D44A, E83A-K84A-E86A,
and
K146A-D149A-E150A mutations to minimize the possibility of
inducing
global misfolding of the proteins (
2,
9). These
proteins
were previously demonstrated to have significant levels
of DNA helicase
activity (
9), indicating specific defects in
functions
required for endonuclease activity. The D40A-D42A-D44A
mutant is also
defective in hairpin DNA binding and self-association
(
9).
Previous mutational analyses suggest that the ability
of Rep68/78 to
form stable oligomers on hairpin DNA is required
for both optimal DNA
binding and
trs endonuclease activity (
9,
51). It
is possible, however, that the endonuclease defects
in the
D40A-D42A-D44A, E83A-K84A-E86A, and K146A-D149A-E150A proteins
could be
the result of disruption of the secondary or tertiary
structure of
regions nearby in either the primary sequence or
the three-dimensional
structure of the protein. The E83A-K84A-E86A
mutation is near a
sequence motif conserved in a superfamily of
replication initiation
proteins which includes Rep68/78 (
17).
This HUHUUU sequence
(in which H represents histidine and U represents
an amino acid with a
bulky hydrophobic side chain) is believed
to be involved in the
coordination of metal ions (
17). The
trs endonuclease activity is known to be dependent on the presence
of
magnesium ions (
13). The sequence in Rep68/78 is HMHVLV
(amino
acids 90 to 95). Mutation of H90 or H92 to alanine has been
shown
previously to knock out the
trs endonuclease activity
of Rep78
on a filled hairpin substrate (
45).
All of our point mutations that resulted in MBP-Rep fusion proteins
which were endonuclease negative on a filled hairpin,
but had
endonuclease levels comparable to that of MBP-Rep68
on
an unfilled
hairpin (Fig.
2), also resulted in proteins which
were helicase
negative (Table
1) (
7,
9,
48,
49). We
hypothesize that many of the mutant Rep68/78 proteins
previously
reported to be endonuclease negative (
7,
9,
24,
28,
48,
49,
51), especially those with mutations outside the
first 200 amino acids of Rep68/78, are endonuclease negative on
a filled hairpin
because of an inability to unwind DNA and not
because of an inability
to cleave at the
trs. Smith and Kotin
(
36) showed
that amino acids 225 to 621 of Rep78 are sufficient
for DNA helicase
activity. Rep68/78 belong to a superfamily of
ATPase/helicase proteins
(
16). All of the sequence motifs conserved
within this
superfamily are found within Rep68/78 amino acids
330 through 422 (
16). The G334A, G339A, K340H, T341A,
E378A-E379A-K381A,
D402A-K404A-K406A, and V418S mutations alter
conserved amino acids
within these helicase motifs.
The D443A-K447A, D455A-D457A, and K463A-E465A-K467A-D468A mutations
alter amino acids within a putative 3,4-heptad repeat
motif which is
believed to be involved in Rep68/78 oligomerization
(
9). The
proteins containing these mutations are endonuclease
negative on a
filled hairpin substrate and helicase negative (Table
1)
(
9). The ability of these mutated proteins to cleave the
unfilled hairpin substrate (Fig.
2) suggests that the proposed
role of
oligomerization, mediated by this motif, in
trs endonuclease
activity (
9) is not required for the actual DNA cleavage
event.
We next wished to determine the minimal portion of the Rep68/78 protein
sufficient for
trs endonuclease activity on an unfilled
hairpin substrate. We made a series of C-terminal truncations
of the
Rep68/78 moiety of MBP-Rep68
. Plasmids encoding C-terminally
truncated versions of MBP-Rep68
were constructed by first amplifying
a section of pMBP-Rep68
(
7) using PCR. Part of each
downstream
primer was complementary to the region where the truncation
was
to be made, and the other part included a
HindIII
site. To make
truncations to Rep68/78 amino acid 150, 200, 251, 300, 352, or
400, the upstream primer was complementary to the region
encoding
Rep68/78 amino acids 3 through 9. To make truncations to
Rep68/78
amino acid 150, 200, 251, or 300, the PCR product was trimmed
with
PstI and
HindIII and used to replace the
PstI-
HindIII fragment
of pMBP-Rep68
, which
contains a portion of the
rep gene. To make
truncations to
amino acid 352 or 400, the PCR product was trimmed
with
BamHI and
HindIII and used to replace the
corresponding
BamHI-
HindIII
fragment of
pMBP-Rep68
.
To construct plasmids encoding MBP fusion proteins truncated to
Rep68/78 amino acid 442, 447, 460, 466, or 476, the upstream
primer was
complementary to the region encoding Rep68/78 amino
acids 357 to 364, which is just upstream of a
SalI site. The PCR
product was
trimmed with
SalI and
HindIII and used to
replace
the
SalI-
HindIII fragment of
pMBP-Rep68
, which contains a portion
of the
rep gene.
Figure
3A shows that endonuclease
activity was detected with a mutant protein containing amino acids 3 to
200 of Rep68/78,
but a protein containing amino acids 3 to 150 had no
detectable
activity on an unfilled hairpin. This confirms that the
first
200 amino acids of Rep68/78 contain all of the essential
components
for cleavage. Our C-terminal truncation mutants which were
truncated
to Rep68/78 amino acid 466 or less lacked the ability to nick
the filled hairpin substrate (Fig.
3B). This is consistent with
previous data using truncated Rep proteins produced in a rabbit
reticulocyte in vitro translation system (
51).
View larger version (94K):
[in this window]
[in a new window]
|
FIG. 3.
Rep68/78 amino acids 3 to 200 are sufficient for
trs endonuclease activity on unfilled hairpin DNA.
trs endonuclease assays were performed (as detailed in the
text) with the truncated fusion proteins indicated above each lane.
Samples contained either no protein, 1.0 µg of MBP-LacZ (LacZ), or
1.0 µg of the fusion protein with the indicated truncation. The
wild-type (W-type) lanes contained 1.0, 0.5, 0.25, or 0.125 µg of
MBP-Rep68. All reaction mixtures were incubated at 37°C for 60 min, boiled, and resolved by nondenaturing PAGE (6% polyacrylamide).
The positions of the substrate and released cleavage product are
indicated on the right. Each sample contained 25,000 cpm of 5'
32P-end-labeled unfilled (A) or filled (B) AAV terminal
repeat hairpin DNA.
|
|
Covalent attachment assays.
Snyder et al. showed that Rep68
can become covalently attached to the 5' phosphate of a T residue at
the nicking site in AAV hairpin DNA and that this linkage involves a
tyrosine residue (37). MBP-Rep68 has also previously been
shown to possess the ability to covalently attach to AAV hairpin DNA
(8). It is unclear whether it is biologically important that
Rep68/78 remain covalently attached to the DNA or if this attachment
simply represents a trapped intermediate in the cleavage process. There
have been reports of Rep78 being tightly associated with virus
particles (21, 29), but no function has been determined for
these attached Rep proteins. In any case, this covalently attaching
tyrosine should be an important component of the endonuclease active site.
Our previous analysis suggested that Y152 is the Rep68/78 active-site
tyrosine (
48). This was based on the observation that
of the
three tyrosines conserved between AAV Rep68/78 and the
human
herpesvirus 6 Rep homologue (
42), the deduced product
of the
only gene known to be able to substitute for the AAV
rep gene in AAV replication (
43), only mutation of Y152
eliminated
the ability of MBP-Rep68
to nick AAV hairpin DNA with a
double-stranded
trs (
48). The Y152F mutant also
was helicase positive, indicating
a specific defect in the endonuclease
function. Tyrosine 156 of
Rep68/78, however, aligns best with the
covalently linking tyrosine
of the NS1 protein of minute virus of mice
(MVM), the only parvovirus
nonstructural protein for which the
active-site tyrosine has been
determined (
27).
In our previous report, we had technical difficulties with the
production of our Y156F mutant (
48). This first batch of
the
Y156F protein had a large amount of a low-molecular-weight
protein
contaminant which apparently interfered with our analysis.
Subsequent
protein preparations from bacteria carrying the same
expression plasmid
also had this low-molecular-weight contaminant.
For the present work,
we reconstructed the plasmid encoding the
Y156F mutant protein. Protein
preparations from bacteria carrying
the reconstructed Y156F plasmid
showed no such contaminant (data
not
shown).
Since it is possible that Y152 and Y156 both participate in the
endonuclease reaction, we also made a double mutant (Y152F/Y156F).
Two
tyrosines separated by three amino acids have been reported
to be
involved in the DNA cleavage reaction performed by the
X174
gene A
protein (
47). It should be noted that the MVM NS1 protein
has a second tyrosine residue right next to the one which forms
the
covalent attachment (
27,
35). We also individually mutated
the other tyrosines in the first 200 amino acids of the Rep68/78
moiety, in the context of the full-length MBP-Rep68
protein.
Plasmids encoding the Y121F, Y152F, and Y175F mutant MBP-Rep proteins
have been reported previously (
48). Plasmids encoding
the
Y5F, Y88F, Y156F, and Y152F/Y156F mutant proteins were made
by an
overlap extension PCR method (
12). Briefly, two pairs
of
primers were used to direct synthesis of mutant DNA fragments.
These
fragments, which overlapped by at least 25 bp, were gel
purified and
subsequently used for overlap extension PCR using
5' and 3' flanking
primers. The resulting amplified product was
gel purified and digested
with appropriate restriction endonucleases,
generating a fragment which
was substituted for the corresponding
fragment within the parent
plasmid, pMBP-Rep68
(
7,
12).
In the cases of the Y5F and
Y88F mutations, a
BglII-
SacII fragment
containing
parts of the
malE (MBP) and
rep genes was
generated.
In the case of the Y156F mutation, a
PstI-
BamHI fragment comprised
of a portion of the
rep gene was generated. The Y152F/Y156F mutation
was made
using the same primers as the Y156F mutation, but the
initial PCR
template was pMBP-Rep68
/Y152F (
48) instead of
pMBP-Rep68
.
PCRs were carried out using the thermostable
Pfu DNA polymerase,
which has a high fidelity (1.3 × 10
6 errors/base pair/cycle) (Stratagene, La Jolla,
Calif.). The presence
of mutations in the
rep coding region
was confirmed by DNA
sequencing.
For our covalent attachment assay, we needed a 3'-end-labeled hairpin
DNA in which the
trs was still single stranded. We therefore
partially filled the AAV hairpin, using only
32P-labeled A
and unlabeled G nucleotides (Fig.
1). This partially
filled hairpin DNA
was then purified by nondenaturing PAGE (6%
polyacrylamide).
Gel-purified, partially filled, 3'
32P-end-labeled AAV
terminal repeat hairpin DNA (25,000 cpm) was
incubated in the presence
of MBP-Rep68
or mutant proteins under
the standard
trs
endonuclease assay conditions. Some samples were
incubated for an
additional 10 min in the presence of 1 µg of
proteinase K at 37°C.
A one-fourth volume of 5× SDS gel-loading
buffer (125 mM Tris-HCl [pH
7.5], 5% [wt/vol] SDS, 50% [vol/vol]
glycerol, 0.25% [wt/vol]
bromophenol blue, 1% [vol/vol]
-mercaptoethanol)
was then added
to each sample. The reaction products were then
boiled for 7 min and
resolved on a 24-cm-long SDS-5% polyacrylamide
gel. The gel was then
soaked for 30 min in a fixing solution containing
20% methanol, 10%
acetic acid, and 5% glycerol; dried; and
autoradiographed.
Figure
4 shows that in the presence of
MBP-Rep68
, there is a radiolabeled species which migrates between
the 147- and 241-kDa
protein standards in SDS-PAGE. This species
migrates slower than
the predominant bands seen with the MBP-LacZ or no
protein negative
controls. This species does not form in the absence of
MgCl
2,
which is known to be required for
trs
endonuclease activity, and
is eliminated by proteinase K treatment.
Based on these observations,
we conclude that this band represents the
previously described
covalent complex between MBP-Rep68
and the
hairpin DNA (
8).
Covalent attachment of MBP-Rep68
to the
partially filled hairpin
did not require ATP. The Y5F, Y88F, Y121F, and
Y175F proteins
covalently attached to the hairpin comparably to the
wild-type
MBP-Rep68
.
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 4.
Covalent attachment assays of wild-type and mutant
MBP-Rep68 fusion proteins. Covalent attachment assays were performed
as detailed in the text. Each sample contained 25,000 cpm of 3'
32P-end-labeled partially filled AAV terminal repeat
hairpin DNA. Samples contained either no protein, 3.0 µg of MBP-LacZ
(LacZ), or 3.0 µg of the mutant fusion protein indicated above each
lane. The descending wild-type (W-type) lanes contained 3.0, 1.5, 0.75, or 0.375 µg of MBP-Rep68. Contents of other lanes are as follows:
W-type w/o ATP, 3.0 µg of MBP-Rep68 in a reaction mixture without
ATP; W-type w/o Mg2+, 3.0 µg of MBP-Rep68 in a
reaction mixture without magnesium; W-type + Proteinase K, 3.0 µg of MBP-Rep68 in a reaction mixtures which was incubated for an
additional 10 min in the presence of 1 µg of proteinase K at 37°C.
Reaction products were resolved by SDS-PAGE (5% polyacrylamide). The
positions of the free substrate and covalently attached product are
indicated on the right. Molecular weight markers are indicated on the
left.
|
|
Only mutation of Y156 completely disrupted the ability of the protein
to covalently attach to the partially filled hairpin
substrate. The
data implicate Y156 as an active-site tyrosine.
The Y152F protein
showed much less covalent attachment than the
wild-type protein. It is
unclear if this means that Y152 can function
as a secondary site of
covalent attachment or if its mutation
disrupts the ability of Y156 to
attach. The nearby K146A-D149A-E150A
mutation also resulted in a
protein that is defective in nicking
(Fig.
2) and therefore is
presumably defective in covalent attachment
as
well.
Endonuclease and helicase assays on the Y156F protein.
We next
wished to examine the trs endonuclease (on unfilled hairpin)
and DNA helicase activities of the Y156F protein. Our previous analysis
(48), with the contaminated Y156F protein preparation,
showed no detectable trs endonuclease (on a filled hairpin
substrate) or DNA helicase activity. The new Y156F protein preparation,
used in the present study, lacks endonuclease activity (Fig.
5A).
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
The Y156F protein lacks trs endonuclease
activity but retains DNA helicase activity. Assays were performed (as
detailed in the text) with MBP-Rep68 (W-type) or the mutant fusion
proteins indicated above each lane. MBP-LacZ (LacZ) was included as a
negative control. Samples contained either no protein or the amount of
each protein (in micrograms) indicated below each lane. (A)
trs endonuclease assays. Each sample contained 25,000 cpm of
5' 32P-end-labeled unfilled AAV hairpin DNA. All reaction
mixtures were incubated at 37°C for 60 min, boiled, and resolved on a
nondenaturing 6% polyacrylamide gel. The positions of the substrate
and released cleavage product are indicated on the right. (B) DNA
helicase assays. Each sample contained 25,000 cpm of
32P-labeled partial duplex substrate. The sample marked
"Boiled" contained no MBP fusion protein and was heated to 100°C
for 5 min. All other reaction mixtures were incubated at 24°C for 35 min and resolved on a nondenaturing 6% polyacrylamide gel. The
positions of the substrate and released 26-mer product are indicated on
the right.
|
|
Helicase assays were performed under the conditions developed by Im and
Muzyczka (
13), with modifications described by
Kyöstiö
and Owens (
22). The DNA helicase
substrate, which consisted
of a radiolabeled 26-mer annealed to
single-stranded M13 DNA,
was prepared as described previously
(
13). The new Y156F protein
preparation has helicase
activity comparable to that of MBP-Rep68
(Fig.
5B), indicating that
the Y156F mutation does not cause a
global disruption of the protein's
structure. The Y152F/Y156F
double mutant performed identically to the
Y156F mutant (Fig.
5).
Elucidation of the mechanism of Rep68/78 endonuclease activity is key
to understanding Rep-mediated preferential integration
and replication
of the AAV genome. The use of partially single-stranded
substrates
should accelerate determination of the portions of
Rep68/78 directly
involved in DNA
cleavage.
| |
ACKNOWLEDGMENTS |
We thank Catherine McKeon, Nancy Nossal, and Robert Craigie for
critical reading of the manuscript. We also thank Ramani Wonderling and
Scotty Walker for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bldg. 8, Rm. 310, 8 Center Dr. MSC 0840, Bethesda, MD 20892-0840. Phone: (301) 496-3359. Fax: (301) 402-0053. E-mail:
ro6n{at}nih.gov.
Present address: Department of Health Administration, City of
Detroit, Detroit, MI 48202.
Present address: Department of Medicine, Louisiana State
University School of Medicine, New Orleans, LA 70112.
| |
REFERENCES |
| 1.
|
Balagué, C.,
M. Kalla, and W. W. Zhang.
1997.
Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.
J. Virol.
71:3299-3306[Abstract].
|
| 2.
|
Bordo, D., and P. Argos.
1991.
Suggestions for "safe" residue substitutions in site-directed mutagenesis.
J. Mol. Biol.
217:721-729[CrossRef][Medline].
|
| 3.
|
Carter, B. J.
1990.
Adeno-associated virus helper functions, p. 255-282.
In
P. Tijssen (ed.), Handbook of parvoviruses, vol. I. CRC Press, Inc., Boca Raton, Fla.
|
| 4.
|
Carter, B. J.,
E. Mendelson, and J. P. Trempe.
1990.
AAV DNA replication, integration, and genetics, p. 169-226.
In
P. Tijssen (ed.), Handbook of parvoviruses, vol. I. CRC Press, Inc., Boca Raton, Fla.
|
| 5.
|
Chejanovsky, N., and B. J. Carter.
1989.
Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication.
Virology
173:120-128[CrossRef][Medline].
|
| 6.
|
Chejanovsky, N., and B. J. Carter.
1989.
Replication of a human parvovirus nonsense mutant in mammalian cells containing an inducible amber suppressor.
Virology
171:239-247[CrossRef][Medline].
|
| 7.
|
Chiorini, J. A.,
M. D. Weitzman,
R. A. Owens,
E. Urcelay,
B. Safer, and R. M. Kotin.
1994.
Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.
J. Virol.
68:797-804[Abstract/Free Full Text].
|
| 8.
|
Chiorini, J. A.,
S. M. Wiener,
R. A. Owens,
S. R. M. Kyöstiö,
R. M. Kotin, and B. Safer.
1994.
Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats.
J. Virol.
68:7448-7457[Abstract/Free Full Text].
|
| 9.
|
Davis, M. D.,
R. S. Wonderling,
S. L. Walker, and R. A. Owens.
1999.
Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro.
J. Virol.
73:2084-2093[Abstract/Free Full Text].
|
| 10.
|
Giraud, C.,
E. Winocour, and K. I. Berns.
1994.
Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence.
Proc. Natl. Acad. Sci. USA
91:10039-10043[Abstract/Free Full Text].
|
| 11.
|
Hauswirth, W. W., and K. I. Berns.
1977.
Origin and termination of adeno-associated virus DNA replication.
Virology
78:488-499[CrossRef][Medline].
|
| 12.
|
Ho, S. N.,
H. D. Hunt,
R. M. Horton,
J. K. Pullen, and L. R. Pease.
1989.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
Gene
77:51-59[CrossRef][Medline].
|
| 13.
|
Im, D. S., and N. Muzyczka.
1990.
The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity.
Cell
61:447-457[CrossRef][Medline].
|
| 14.
|
Im, D. S., and N. Muzyczka.
1989.
Factors that bind to adeno-associated virus terminal repeats.
J. Virol.
63:3095-3104[Abstract/Free Full Text].
|
| 15.
|
Im, D. S., and N. Muzyczka.
1992.
Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization.
J. Virol.
66:1119-1128[Abstract/Free Full Text].
|
| 16.
|
Koonin, E. V.
1993.
A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.
Nucleic Acids Res.
21:2541-2547[Abstract/Free Full Text].
|
| 17.
|
Koonin, E. V., and T. V. Ilyina.
1993.
Computer-assisted dissection of rolling circle DNA replication.
BioSystems
30:241-268[CrossRef][Medline].
|
| 18.
|
Kotin, R. M.,
R. M. Linden, and K. I. Berns.
1992.
Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination.
EMBO J.
11:5071-5078[Medline].
|
| 19.
|
Kotin, R. M.,
J. C. Menninger,
D. C. Ward, and K. I. Berns.
1991.
Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter.
Genomics
10:831-834[CrossRef][Medline].
|
| 20.
|
Kotin, R. M.,
M. Siniscalco,
R. J. Samulski,
X. Zhu,
L. Hunter,
C. A. Laughlin,
S. McLaughlin,
N. Muzyczka,
M. Rocchi, and K. I. Berns.
1990.
Site-specific integration by adeno-associated virus.
Proc. Natl. Acad. Sci. USA
87:2211-2215[Abstract/Free Full Text].
|
| 21.
|
Kube, D. M.,
S. Ponnazhagan, and A. Srivastava.
1997.
Encapsidation of adeno-associated virus type 2 Rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells.
J. Virol.
71:7361-7371[Abstract].
|
| 22.
|
Kyöstiö, S. R. M., and R. A. Owens.
1996.
Identification of mutant adeno-associated virus Rep proteins which are dominant-negative for DNA helicase activity.
Biochem. Biophys. Res. Commun.
220:294-299[CrossRef][Medline].
|
| 23.
|
Linden, R. M.,
E. Winocour, and K. I. Berns.
1996.
The recombination signals for adeno-associated virus site-specific integration.
Proc. Natl. Acad. Sci. USA
93:7966-7972[Abstract/Free Full Text].
|
| 24.
|
McCarty, D. M.,
T. H. Ni, and N. Muzyczka.
1992.
Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro.
J. Virol.
66:4050-4057[Abstract/Free Full Text].
|
| 25.
|
McCarty, D. M.,
J. H. Ryan,
S. Zolotukhin,
X. Zhou, and N. Muzyczka.
1994.
Interaction of the adeno-associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat.
J. Virol.
68:4998-5006[Abstract/Free Full Text].
|
| 26.
|
Mendelson, E.,
J. P. Trempe, and B. J. Carter.
1986.
Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide.
J. Virol.
60:823-832[Abstract/Free Full Text].
|
| 27.
|
Nüesch, J. P. F.,
S. F. Cotmore, and P. Tattersall.
1995.
Sequence motifs in the replicator protein of parvovirus MVM essential for nicking and covalent attachment to the viral origin: identification of the linking tyrosine.
Virology
209:122-135[CrossRef][Medline].
|
| 28.
|
Owens, R. A.,
M. D. Weitzman,
S. R. M. Kyöstiö, and B. J. Carter.
1993.
Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins.
J. Virol.
67:997-1005[Abstract/Free Full Text].
|
| 29.
|
Prasad, K. M. R., and J. P. Trempe.
1995.
The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion.
Virology
214:360-370[CrossRef][Medline].
|
| 30.
|
Ryan, J. H.,
S. Zolotukhin, and N. Muzyczka.
1996.
Sequence requirements for binding of Rep68 to the adeno-associated virus terminal repeats.
J. Virol.
70:1542-1553[Abstract].
|
| 31.
|
Samulski, R. J.,
L. S. Chang, and T. Shenk.
1987.
A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.
J. Virol.
61:3096-3101[Abstract/Free Full Text].
|
| 32.
|
Samulski, R. J.,
A. Srivastava,
K. I. Berns, and N. Muzyczka.
1983.
Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV.
Cell
33:135-143[CrossRef][Medline].
|
| 33.
|
Senapathy, P.,
J. D. Tratschin, and B. J. Carter.
1984.
Replication of adeno-associated virus DNA. Complementation of naturally occurring rep mutants by a wild-type genome or an ori mutant and correction of terminal palindrome deletions.
J. Mol. Biol.
179:1-20[CrossRef][Medline].
|
| 34.
|
Shelling, A. N., and M. G. Smith.
1994.
Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene.
Gene Ther.
1:165-169[Medline].
|
| 35.
|
Skiadopoulos, M. H., and E. A. Faust.
1993.
Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice.
Virology
194:509-517[CrossRef][Medline].
|
| 36.
|
Smith, R. H., and R. M. Kotin.
1998.
The Rep52 gene product of adeno-associated virus is a DNA helicase with 3'-to-5' polarity.
J. Virol.
72:4874-4881[Abstract/Free Full Text].
|
| 37.
|
Snyder, R. O.,
D. S. Im, and N. Muzyczka.
1990.
Evidence for covalent attachment of the adeno-associated virus (AAV) Rep protein to the ends of the AAV genome.
J. Virol.
64:6204-6213[Abstract/Free Full Text].
|
| 38.
|
Snyder, R. O.,
D. S. Im,
T. Ni,
X. Xiao,
R. J. Samulski, and N. Muzyczka.
1993.
Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein.
J. Virol.
67:6096-6104[Abstract/Free Full Text].
|
| 39.
|
Snyder, R. O.,
R. J. Samulski, and N. Muzyczka.
1990.
In vitro resolution of covalently joined AAV chromosome ends.
Cell
60:105-113[CrossRef][Medline].
|
| 40.
|
Srivastava, A.,
E. W. Lusby, and K. I. Berns.
1983.
Nucleotide sequence and organization of the adeno-associated virus 2 genome.
J. Virol.
45:555-564[Abstract/Free Full Text].
|
| 41.
|
Straus, S. E.,
E. D. Sebring, and J. A. Rose.
1976.
Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis.
Proc. Natl. Acad. Sci. USA
73:742-746[Abstract/Free Full Text].
|
| 42.
|
Thomson, B. J.,
S. Efstathiou, and R. W. Honess.
1991.
Acquisition of the human adeno-associated virus type-2 rep gene by human herpesvirus type-6.
Nature (London)
351:78-80[CrossRef][Medline].
|
| 43.
|
Thomson, B. J.,
F. W. Weindler,
D. Gray,
V. Schwaab, and R. Heilbronn.
1994.
Human herpesvirus 6 (HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2) and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression.
Virology
204:304-311[CrossRef][Medline].
|
| 44.
|
Trempe, J. P.,
E. Mendelson, and B. J. Carter.
1987.
Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli.
Virology
161:18-28[CrossRef][Medline].
|
| 45.
|
Urabe, M.,
Y. Hasumi,
A. Kume,
R. T. Surosky,
G. J. Kurtzman,
K. Tobita, and K. Ozawa.
1999.
Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein.
J. Virol.
73:2682-2693[Abstract/Free Full Text].
|
| 46.
|
Urcelay, E.,
P. Ward,
S. M. Wiener,
B. Safer, and R. M. Kotin.
1995.
Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein.
J. Virol.
69:2038-2046[Abstract].
|
| 47.
|
van Mansfeld, A. D. M.,
H. A. A. M. van Teeffelen,
P. D. Baas, and H. S. Jansz.
1986.
Two juxtaposed tyrosyl-OH groups participate in X174 gene A protein catalysed cleavage and ligation of DNA.
Nucleic Acids Res.
14:4229-4238[Abstract/Free Full Text].
|
| 48.
|
Walker, S. L.,
R. S. Wonderling, and R. A. Owens.
1997.
Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity.
J. Virol.
71:2722-2730[Abstract].
|
| 49.
|
Walker, S. L.,
R. S. Wonderling, and R. A. Owens.
1997.
Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.
J. Virol.
71:6996-7004[Abstract].
|
| 50.
|
Ward, P.,
E. Urcelay,
R. Kotin,
B. Safer, and K. I. Berns.
1994.
Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.
J. Virol.
68:6029-6037[Abstract/Free Full Text].
|
| 51.
|
Weitzman, M. D.,
S. R. M. Kyöstiö,
B. J. Carter, and R. A. Owens.
1996.
Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA.
J. Virol.
70:2440-2448[Abstract].
|
| 52.
|
Weitzman, M. D.,
S. R. M. Kyöstiö,
R. M. Kotin, and R. A. Owens.
1994.
Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.
Proc. Natl. Acad. Sci. USA
91:5808-5812[Abstract/Free Full Text].
|
| 53.
|
Wonderling, R. S.,
S. R. M. Kyöstiö, and R. A. Owens.
1995.
A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.
J. Virol.
69:3542-3548[Abstract].
|
| 54.
|
Wonderling, R. S., and R. A. Owens.
1997.
Binding sites for adeno-associated virus Rep proteins within the human genome.
J. Virol.
71:2528-2534[Abstract].
|
| 55.
|
Wu, J.,
M. D. Davis, and R. A. Owens.
1999.
Factors affecting the terminal resolution site endonuclease, helicase and ATPase activities of adeno-associated virus type 2 Rep proteins.
J. Virol.
73:8235-8244[Abstract/Free Full Text].
|
| 56.
|
Zhou, X.,
I. Zolotukhin,
D. S. Im, and N. Muzyczka.
1999.
Biochemical characterization of adeno-associated virus Rep68 DNA helicase and ATPase activities.
J. Virol.
73:1580-1590[Abstract/Free Full Text].
|
Journal of Virology, March 2000, p. 2936-2942, Vol. 74, No. 6
0022-538X/00/$04.00+0
This article has been cited by other articles:
-
van Nierop, G. P., de Vries, A. A. F., Holkers, M., Vrijsen, K. R., Goncalves, M. A. F. V.
(2009). Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease. Nucleic Acids Res
37: 5725-5736
[Abstract]
[Full Text]
-
Hewitt, F. C., Li, C., Gray, S. J., Cockrell, S., Washburn, M., Samulski, R. J.
(2009). Reducing the Risk of Adeno-Associated Virus (AAV) Vector Mobilization with AAV Type 5 Vectors. J. Virol.
83: 3919-3929
[Abstract]
[Full Text]
-
Jang, M. Y., Yarborough, O. H. III, Conyers, G. B., McPhie, P., Owens, R. A.
(2005). Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19. J. Virol.
79: 3544-3556
[Abstract]
[Full Text]
-
Yoon-Robarts, M., Blouin, A. G., Bleker, S., Kleinschmidt, J. A., Aggarwal, A. K., Escalante, C. R., Linden, R. M.
(2004). Residues within the B' Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2. J. Biol. Chem.
279: 50472-50481
[Abstract]
[Full Text]
-
James, J. A., Aggarwal, A. K., Linden, R. M., Escalante, C. R.
(2004). Structure of adeno-associated virus type 2 Rep40-ADP complex: Insight into nucleotide recognition and catalysis by superfamily 3 helicases. Proc. Natl. Acad. Sci. USA
101: 12455-12460
[Abstract]
[Full Text]
-
Schmidt, M., Katano, H., Bossis, I., Chiorini, J. A.
(2004). Cloning and Characterization of a Bovine Adeno-Associated Virus. J. Virol.
78: 6509-6516
[Abstract]
[Full Text]
-
Bossis, I., Chiorini, J. A.
(2003). Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles. J. Virol.
77: 6799-6810
[Abstract]
[Full Text]
-
Yoon-Robarts, M., Linden, R. M.
(2003). Identification of Active Site Residues of the Adeno-associated Virus Type 2 Rep Endonuclease. J. Biol. Chem.
278: 4912-4918
[Abstract]
[Full Text]
-
Yoon, M., Smith, D. H., Ward, P., Medrano, F. J., Aggarwal, A. K., Linden, R. M.
(2001). Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro. J. Virol.
75: 3230-3239
[Abstract]
[Full Text]
Top
Abstract
Text
