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Previous Article | Next Article 
Journal of Virology, March 2000, p. 2936-2942, Vol. 74, No. 6
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
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.
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 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 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
0022-538X/00/$04.00+0
Mutational Analysis of Adeno-Associated Virus Type
2 Rep68 Protein Endonuclease Activity on Partially
Single-Stranded Substrates
and
ABSTRACT
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, 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.
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.
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 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.
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 (M225dl) was made by replacing the PstI-BamHI fragment of the rep gene of
pMBP-Rep68 with the corresponding fragment from
pHIV-Rep/M225dl (28).
Figure 2 shows that the Y121F,
M225dl, 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.
|
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.
|
. 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).
|
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.
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
MgCl2, 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.
|
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).
|
(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.
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