From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain
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ABSTRACT |
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Initiation of phage DNA replication is a semiconservative process in which a DNA
polymerase uses one DNA strand as a template for the synthesis of its
complementary strand. All DNA polymerases require a preexisting primer
to initiate DNA synthesis (1). In many cases, the primer is a short RNA
or DNA molecule. In linear DNAs, which are unable to form circular or
hairpin structures during replication, replication of the genome ends
cannot take place via RNA or DNA priming. Several mechanisms have
evolved to solve the problem of initiation of DNA replication in such
linear genomes (2). In the case of viral linear genomes, the OH group
of a serine, threonine, or tyrosine residue of a terminal protein
(TP)1 molecule serves as a
primer for DNA replication, and as a consequence, the TP molecule
becomes covalently attached to the 5'-terminal nucleotide. This
mechanism of initiation of replication is called protein priming
(reviewed in Ref. 3). Once bound to the DNA, the TP may serve
additional functions such as assisting in DNA packaging (4, 5),
protection against nucleases (6), enhancement of infectivity (7, 8),
stimulation of template activity (9), matrix attachment (10),
stimulation of transcription (10), and recruitment of a new TP to the
origin (11). As a step previous to the initiation of replication, TP
interacts with the DNA polymerase to form a heterodimer (12). To
distinguish between the different functions of TP, we will refer to the
TP bound to the DNA ends as parental TP and the TP in the heterodimer with the DNA polymerase as primer TP. Interaction of primer TP and/or
DNA polymerase with parental TP may increase the affinity of the TP/DNA
polymerase heterodimer for the origin or could assist the latter for
its correct positioning at the origin, which may be important for
initiation of DNA replication at the correct position.
The protein-priming mechanism of DNA replication has been mainly
studied in the Bacillus subtilis bacteriophage Structure-function studies and biochemical characterization of DNA, Proteins, and Chemicals--
[ Amino Acid Sequence Comparison--
Multiple alignment of the TP
sequences was performed in two steps. The selected protein sequences
were aligned with the GAP program (27). Then, the alignments obtained
were adjusted manually to find generally conserved regions or residues
colinearly arranged.
Site-directed Mutagenesis and Expression of Psoralen Cross-linking and Spreading of DNA Molecules for
Electron Microscopy--
To analyze the structure of replicative
intermediates produced during Single Substitutions at Residues Lys61,
Asn80, Tyr82, Gly83, and
Ser87 in
To study the function of the most conserved residues in the TP region
from position 61 to 87, single changes were introduced in two
Replication of
To determine whether the presence of parental TP affects the primer
function of the mutant TPs, the in vitro formation of the
TP-dAMP initiation complex using
After the formation of the TP-dAMP initiation complex, the DNA
polymerase remains associated with the primer TP until a short DNA
primer of 9 nucleotides has been formed. It is only after this
so-called transition step that the two proteins dissociate, and the DNA
polymerase continues elongation until complete replication of the
nascent DNA chain is achieved (35). To study possible effects of the
mutations introduced in the TP on the transition step, we carried out
replication assays in which one full round of replication is allowed
(see "Materials and Methods"). As shown in Fig.
2, the amount of replicated DNA and the
velocity of the reactions were similar when either wild-type or mutant
TPs was used (see also Table I). Therefore, the single amino acid
changes introduced in the
These results suggest that the mutant TPs are able to serve as a primer
with an efficiency comparable to that of wild-type TP indicating that
the residues Lys61, Asn80, Tyr82,
Gly83, and Ser87 are not involved in the primer
activity of the TP, and they are not involved in the transition step.
Mutations at the Asn80 and Tyr82 Residues
Alter the Parental TP Function--
An in vitro Detection of Single-stranded DNA Molecules as a Consequence of the
Inactivation of the Replication Origins--
The explanation depicted
in Fig. 5B predicts the generation of full-size displaced
ssDNA. To detect the presence of these ssDNA molecules, samples of the
amplification assays (after 40 min) were subjected to electron
microscopy analysis (see "Materials and Methods"). To distinguish
between dsDNA and ssDNA molecules, a psoralen cross-linking procedure
was used (30). It has been shown that psoralen treatment of
Previous analysis of the different types of replicative intermediate
molecules synthesized during Addition of Wild-type TP/DNA Polymerase Heterodimer Counteracts the
Inactive Origin--
The above results show that mutations at residues
N80 or Y82 of Conclusion--
Single substitutions at conserved residues
Lys61, Asn80, Tyr82,
Gly83, and Ser87 of
Altogether, the results presented in this paper indicate that
recognition of 29 DNA replication starts
with the recognition of the origin of replication, located at both ends
of the linear DNA, by a heterodimer formed by the
29 terminal
protein (TP) and the
29 DNA polymerase. The parental TP, covalently
linked to the DNA ends, is one of the main components of the
replication origin. Here we provide evidence that recognition of the
origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on
amino acid sequence comparisons, various
29 TP mutants were
generated at conserved amino acid residues from positions 61 to 87. In vitro
29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for
functional interaction between primer and parental TP required for
recognition of the origin of replication. Although these mutant TPs can
form functional heterodimers with
29 DNA polymerase that are able to
recognize the origin of replication, these heterodimers are not able to
recognize an origin containing a mutant TP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
29 and
adenoviruses (see Refs. 2, 3, 13, and 14 for review). The
29 genome is a 19,285-base pair linear double-stranded DNA molecule (15) with a
phage-encoded 31-kDa TP covalently attached to the 5' termini (16).
Genetic studies and the development of an in vitro DNA replication system (17) led to the identification of the origins of
replication at each end of the DNA molecule (18, 19). To activate the
initiation of replication, dsDNA-binding protein p6 forms a
nucleoprotein complex that would help to open the DNA ends (20). A
primer TP/DNA polymerase heterodimer recognizes the origin of
replication, probably through protein-protein interaction of the primer
TP and/or
29 DNA polymerase with the parental TP. Then, the
29
DNA polymerase catalyzes the addition of the first dAMP (21) to the OH
group provided by Ser232 of the primer TP (22). The
formation of this first TP-dAMP is directed by the second nucleotide at
the 3'-end of the template and then slides back one nucleotide to
recover the terminal nucleotide (23). Following initiation, the same
29 DNA polymerase molecule completes replication of the parental strand.
29 parental TP is an important requirement for in vitro
initiation of
29 DNA replication (24). Two lines of evidence show that parental TP is also required in vivo. First, in a mixed
infection experiment at 42 °C using phages that have a
thermosensitive (ts) mutation in either gene 2 (encoding the
DNA polymerase) or gene 3 (encoding TP), most of the phage progeny had
the ts2 genotype (16), and second, successful transfection
required an intact gene 3 product (17, 25). Moreover, formation of the
TP-dAMP initiation complex was obtained using as template the TP-DNA
isolated from the closely related Bacillus phage ø15 but
not from the more distantly related Bacillus phage GA-1 or
the pneumococcal phage Cp-1. The lack of activity in the initiation
reaction of the TP-DNA isolated from these latter two phages could be
because of differences in the parental TPs (24).
29 TP
provide a basis to gain insight about the different roles of this
protein in
29 DNA replication. As shown here, a mutational analysis
of
29 TP at conserved amino acids from positions 61 to 87 revealed
that residues Asn80 and Tyr82 are important for
recognition of the origin of replication. The results obtained indicate
that parental TP plays an important role as a structural part of the
replication origin through functional interactions with the primer
TP.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-32P]dATP
(3000 Ci/mmol) and unlabeled nucleotides were obtained from Amersham
Pharmacia Biotech. TP-DNA was obtained as described (17). Proteinase K
and DNA primers were from Roche Molecular Biochemicals. Reagents and
materials for electron microscopy were from Balzers except
benzyldimethylalkylammonium chloride, which was purchased from
Bayer. Wild-type and mutant
29 TPs were purified as described by
Zaballos et al. (26).
29 TP
Mutants--
The wild-type
29 TP gene, cloned into M13mp18, was
amplified using the primer 5'-GAC ATC GAA TTC TAT TCA GAA GTT G-3'
(sense) and 5'-CAT ATG CTG GAT CCT TTA ACG GAG C-3' (antisense). The
site-directed mutagenesis was carried out by polymerase chain reaction
using the primer 5'-GAC GCT TGT TCC ATC CAC TTA TTG-3' for mutant K61M, 5'-GAC GCT TGT TCC CTC CAC TTA TTG-3' for K61R, 5'-GAC GCT TGT TCC GTC
CAC TTA TTG-3' for K61T, 5'-CAC ACC GTA TGC CCT CTT TTC GAA C-3' for
N80S, 5'-CAC CAC ACC TAA TGC ATT CTT TTC G-3' for Y82L, 5'-CAC CAC ACC
GGA TGC ATT CTT TTC G-3' for Y82S, 5'-CTA GCC ACC ACA TCG TAT GCA
TTC-3' for G83D, and 5'-CTT AGC TTT ACT AGC CAC CAC ACC G-3' for S87R.
The polymerase chain reaction fragments carrying the different
mutations were subcloned in plasmid pAZe3s (28), which expresses
29
TP under the control of the
PL promoter. The presence
of the desired mutations and the absence of additional mutations were
confirmed by sequencing each
29 TP mutant gene using the primers:
5'-GAC ATC ACG TTC TAT TC-3', 5'-GGA AGG AAC AAG CGT CC-3', 5'-GAC CCC
ATG ATT TTG AC-3', and 5'-GAA TTT GAT AGT GAG GG-3'. Sequencing was
carried out by the chain termination method (29), using Sequenase kit
version 2.0 from U. S. Biochemical Corp. Expression of the mutant
proteins was carried out in the Escherichia coli
strain NF1.
29 TP-dAMP Formation (Protein-primed Initiation
Assay)--
The incubation mixtures contained (in 25 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 20 mM ammonium sulfate, 4%
glycerol, 0.1 mg/ml bovine serum albumin, 0.25 µM
[
-32P] dATP (2.5 µCi), 0.5 µg of
29 TP-DNA, or
0.2 µg of single-stranded 29-mer oligonucleotide with the sequence
corresponding to the right
29 DNA end (oriR(29), 5'-AAA GTA GGG TAC
AGC GAC AAC ATA CAC CA-3'), 10 ng of wild-type or mutant
29 TP, and
20 ng
29 DNA polymerase. When indicated, oriR(29) was used as a
template instead of TP-DNA, and 1 mM MnCl2 was
used as a metal activator. After incubation for the indicated time at
30 °C (in conditions shown to be linear with time and enzyme
amount), the reactions were stopped by adding EDTA to 10 mM
and SDS to 0.1%. The samples were filtered through Sephadex G-50 spin
columns in the presence of 0.1% SDS. The excluded volume was analyzed
by SDS-polyacrylamide gel electrophoresis and autoradiography. The
relative amounts of incorporated [
32-P]dAMP were
calculated by densitometric analysis of the autoradiographs.
29 TP-DNA Replication Assay (Protein-primed Initiation Plus
Elongation)--
The incubation mixtures contained (in 25 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
20 mM ammonium sulfate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 0.5 µg of TP-DNA, 20 µM each dCTP, dGTP, dTTP, and [
-32P]dATP
(1 µCi), 10 ng of wild-type or mutant TP, and 20 ng of
29 DNA
polymerase. After incubation for the indicated time at 30 °C, the
reactions were stopped, and the samples were filtered as described in
the previous paragraph. Quantitation of the DNA synthesized in
vitro was carried out from the amount of radioactivity (Cerenkov
radiation) corresponding to the excluded volume. The size of the
synthesized DNA was determined by alkaline agarose gel electrophoresis
followed by autoradiography.
29 TP-DNA Amplification Assay--
The incubation mixtures
contained (in 10 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM ammonium sulfate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum
albumin, 25 ng of TP-DNA, 80 µM each dCTP, dGTP, dTTP and
[
-32P] dATP (1 µCi), 8 µg of protein p5 (ssDNA
binding), 10 µg of protein p6, 20 ng of
29 DNA polymerase, and 10 ng of either wild-type or mutant TP. After incubation for 1.5 h at
30 °C, or at the indicated times, the reactions were stopped and the
samples were filtered as described above. The DNA amplification factor
was calculated as the ratio between the amount of DNA at the end of the
reaction (input DNA plus synthesized DNA) and the amount of input
29
DNA. Quantitation of the DNA synthesized in vitro was
carried out from the amount of radioactivity (Cerenkov radiation)
measured from the excluded volume (2.66 nmol of dNTP incorporated,
which correspond to 0.93 × 106 cpm, allows the
synthesis of 887 ng of dsDNA). When indicated, the size of the
amplified DNA was analyzed by either alkaline or native agarose gel
electrophoresis followed by autoradiography and ethidium bromide staining.
29 DNA amplification in
vitro, amplification reactions were stopped by adding 0.05 volume
of 4,5',8-trimethylpsoralen (200 µg/ml in 100% ethanol), and the
samples were maintained on ice for 5 min in the dark. Then, they were
irradiated with 360 nm of ultraviolet light on ice for 30 min as
described by Sogo and Thoma (30). After psoralen cross-linking, the
samples were digested with proteinase K (500 µg/ml) for 2 h at
65 °C and extracted with phenol, and the DNA was precipitated with
ethanol. Denaturation and spreading of the psoralen-cross-linked DNA
for electron microscopy were carried out according to the
benzyldimethylalkylammonium chloride technique as described by Sogo
et al. (31). Electron micrographs were taken routinely at 80 kV at a magnification of × 10,000. Contour length measurements
were carried out on photographic prints using a Summagraphic digitizer tablet.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
29 TP Do Not Affect Its Function as a
Primer--
A multiple alignment of the amino acid sequence of TPs of
Bacillus sp. phages
29, PZA, Nf, B103 and GA-1, and TP of
the E. coli phage PRD1 is presented in Fig.
1. These TPs are very similar in size,
ranging from 258 (phage PRD1) to 267 (phage Nf) amino acids. The
percentage of identical amino acids shared by these TPs is 9% (22%
similarity) increasing to 32% (52% similarity) when only the TPs of
Bacillus sp. are considered. Nonetheless, the TPs from the
Bacillus sp. phages and from the E. coli phage PRD1 share a significant number of conserved amino acids, some of which
may be important for its protein structure and/or function.
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Fig. 1.
Multiple alignment of the TP amino acid
sequences of Bacillus sp. and E. coli bacteriophages. EMBL data base accession number
are given in parentheses: 29 (J02479), PZA (M11813), Nf (Y00363),
B103 (X99260), GA1 (X96987), and the E. coli bacteriophage
PRD1 (M22161). Numbers at the beginning of the amino acid
sequence refer to the position in the protein sequence. Black
boxes enclose residues conserved in all sequences compared, and
gray boxes enclose residues that are only conserved in the
TPs of Bacillus bacteriophages. The following amino acids
are considered conservative: S and T; A and G; K and R; D, E, Q, and N;
I, L, M, V, Y, and F. The residues of
29 TP that have been subjected
to mutagenesis are indicated with an asterisk.
29 TP
residues that are invariant in all the aligned TPs shown in Fig. 1
(Lys61 and Ser87) and in three additional
residues that are invariant in the TPs of the Bacillus
phages (Asn80, Tyr82, and Gly83).
The changes were designed taking into account secondary structure predictions (32, 33) and general suggestions for conservative substitutions (34). Eight mutants were obtained K61M, K61R, K61T, N80S,
Y82L, Y82S, G83D, and S87R. Site-directed mutagenesis, overproduction,
and purification of the mutant proteins were carried out as described
under "Materials and Methods."
29 TP-DNA starts at both ends by a specific
protein-priming mechanism. In this process the viral DNA polymerase, which forms a heterodimer with the
29 TP, catalyzes the linkage of
dAMP to the TP, which acts as a primer. To evaluate the primer function
of mutant TPs, we studied the formation of the TP-dAMP initiation
complex (initiation reaction) using as a template a single-stranded
29-mer oligonucleotide with the sequence corresponding to the right
29 DNA end (oriR(29)) but lacking parental TP (see "Materials and
Methods"). As shown in Table I, the
amount of dAMP incorporated to the various mutant TPs was comparable to that of the wild-type TP, indicating that these mutant TPs interact with
29 DNA polymerase and that they are able to serve as a primer in a reaction that does not involve interactions with parental TP.
Functions of wild-type and mutant ø29 TPs
29 TP-DNA as a template was assayed
with wild-type or the mutant TPs. The results were similar to those
obtained when an oligonucleotide was used as a template (see Table I).
Therefore, the interactions between the mutant TP/DNA polymerase
heterodimer and the wild-type parental TP, which probably take place
during the recognition of the origin in the initiation of replication,
are not affected by these mutations.
29 TP do not affect the dissociation of
the
29 TP/DNA polymerase heterodimer needed to proceed into
elongation in the DNA replication process.
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Fig. 2.
In vitro 29 TP-DNA
replication. The assays were carried out in the presence of 10 ng
of either
29 wild-type (wt) or mutant TPs and 20 ng of
29 DNA polymerase (see "Materials and Methods"). After
incubation for the indicated times at 30 °C, the samples were first
used to calculate their relative replication activity by measuring the
amount of incorporated [
-32P]dAMP (see Table I). Next,
the samples were run in an alkaline agarose gel to determine the
lengths of the synthesized DNAs. The migration position of unit length
29 DNA is indicated. bp, base pairs.
29 DNA
amplification system has been described (36) that requires the
following purified proteins of
29: TP, DNA polymerase, the
dsDNA-binding protein p6, and the ssDNA-binding protein p5. During the
first round of
29 TP-DNA amplification the TP present in the TP/DNA
polymerase heterodimer acts as a primer and becomes covalently linked
to the newly synthesized DNA strand. In subsequent replication rounds
this TP molecule will act as a parental TP. This in vitro
assay mimics the natural amplification of viral DNA. To analyze the
possible effects of the mutations introduced in the TP on its function
as parental TP, DNA amplification assays were carried out as described
under "Materials and Methods." Fig. 3
shows the amount of DNA synthesized using wild-type or mutant TPs.
Mutants K61M, K61R, K61T, G83D, and S87R led to levels of amplification
similar or even higher than wild-type TP (see also Table I). However,
mutants N80S, Y82L, and Y82S were very inefficient in the amplification
reactions despite the fact that they have wild-type-like primer
activity. To determine in which stage of the amplification process
these mutant TPs are affected, the synthesis of DNA was studied as a function of time. DNA amplification experiments were carried out as
described above with wild-type and mutant TPs N80S, Y82L, or Y82S. As
shown in Fig. 4, the amount of DNA
synthesized increased during the first 10 min, using as primer either
the wild-type or mutant TPs. Afterward, DNA synthesis primed by the
wild-type TP continued increasing with time, whereas DNA synthesis
primed by mutant TPs begins to reach a plateau after 20 min. Analysis by native agarose gel electrophoresis showed that, in all cases, unit
length
29 DNA was synthesized (Fig. 4B). The amount of
de novo synthesized DNA (in nanograms) was quantified from
the amount of radioactivity incorporated into DNA (Fig. 4A),
and the amplification factor was calculated as the ratio between the
amount of DNA at the end of the reaction (input DNA plus synthesized
DNA) and the amount of input DNA. The data, shown in Table
II, indicate that the amount of de
novo synthesized DNA initiated with these mutant TPs implies an
amplification factor close to 3 and never exceeded this value even
after extended incubation times. Assuming that all DNA molecules were
initiated and replicated completely, a 2-fold amplification factor is
expected after the first round of replication in a normal process,
becoming a 4-fold amplification factor after completion of the second
round (see Fig. 5A). Control experiments with the wild-type TP showed amplification levels close to
30-fold (Table II). However, with mutant TPs N80S, Y82L, or Y82S the
amplification factor only reached a maximum of 3, indicating that DNA
synthesis was stalled in some way before completing a second round of
replication. These results are explained in the scheme shown in Fig.
5B. After a wild-type TP-DNA molecule has been replicated in
an in vitro experiment containing a mutant TP in the
reaction mixture, both daughter molecules generated contain one origin
of replication with a wild-type TP and the other with a mutant TP. If
the presence of the mutant TP inactivates the corresponding origin, the
second round of replication will be 50% productive because only the
origin with a wild-type TP could be used for initiation. Therefore, if
this model is correct, 3-fold is the maximal amplification factor
expected in the assay. This is in agreement with the data obtained in
the quantitation of the DNA synthesized in vitro (see Table
II). Moreover, replication would stop after this incomplete second
replication round because now all double-stranded origins would contain
a mutant TP, being the DNA strands corresponding to the input DNA fully
displaced. The possibility that the two displaced parental strands
could hybridize to reconstitute a molecule with two active origins is precluded by the presence of
29 ssDNA- binding protein in the assay.
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Fig. 3.
In vitro 29 TP-DNA
amplification with
29 wild-type or mutant
TPs. The
29 TP-DNA amplification assays were carried out as
described under "Materials and Methods," in the presence of 20 ng
of
29 DNA polymerase and 10 ng of the indicated TP. The amount and
size of amplified DNA was analyzed by alkaline agarose gel
electrophoresis followed by ethidium bromide staining. The amount of
input DNA is shown as control (C). wt,
wild-type.
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Fig. 4.
Kinetics of in vitro
29 TP-DNA amplification with
29 wild-type or mutant TPs N80S, Y82L, or
Y82S. A, DNA synthesized in vitro as a
function of time. The amplification assay was carried out as described
under "Materials and Methods," in the presence of 25 ng of
29
TP-DNA as template, 10 ng of
29 wild-type TP, or mutants N80S, Y82L,
or Y82S. After incubation for the indicated times at 30 °C, the
reaction was stopped and quantitated as the total amount (in nanograms)
of dNTP incorporated, and (B) the sizes of the synthesized
DNAs were analyzed by native agarose gel electrophoresis. The migration
position of unit length
29 DNA is indicated. wt, wild
type; bp, base pairs.
Kinetic analysis of in vitro ø29 DNA amplification
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Fig. 5.
Model for in vitro
29 DNA amplification. Schematic
29 DNA
amplification with wild-type TP (A) or mutant TPs N80S,
Y82L, or Y82S (B). The different replicative intermediates
produced during the first DNA replication round are depicted. Wild-type
and mutant TPs are indicated in white and black,
respectively. Bold lines represent the newly synthesized DNA
strands, and plain lines represent parental DNA strands.
RI, replicative intermediate.
29 DNA
in the presence of protein p6 followed by short UV light irradiation
and DNA spreading under denaturing conditions produces molecules that
contain small bubbles corresponding to ssDNA regions, in which the
binding of p6 prevents psoralen cross-linking (37). Taking into account
that protein p6 binds to dsDNA and not to ssDNA and that
29
ssDNA-binding protein covers the displaced strand of the replicative
intermediate, molecules without bubbles correspond to displaced ssDNA.
Fig. 6A shows a typical
example of a replicative intermediate taken from a DNA amplification
assay using the wild-type TP, and it consists of a dsDNA with one ssDNA
branch that corresponds to the displaced strand from one origin. In
addition to molecules with a single ssDNA branch, DNA molecules were
observed with two ssDNA branches. However, no full-length
29 ssDNA
molecules were observed in these samples (results not shown). Electron
microscopy analysis of DNA amplification products using mutant TPs
N80S, Y82L, or Y82S revealed the appearance of full-length
29 ssDNA molecules as shown in Fig. 6B for the mutant TP Y82L.
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Fig. 6.
Electron microscopy of in vitro
cross-linked 29 DNA amplified either
with wild-type TP (A) or with mutant Y82L TP
(B). Samples were treated with psoralen and
spread as described under "Materials and Methods." Denatured pUC18
DNA was used as a size marker. Short and long
arrows point to dsDNA and ssDNA regions, respectively. Note that
B shows a full-length
29 ssDNA molecule. Bars
correspond to 500 base pairs.
29 DNA replication indicated that
neither in vivo (18) nor in vitro (38)
full-length
29 ssDNA molecules were detected when natural
29 DNA
template with TP at both DNA ends was used. Full-length ssDNA molecules
were only observed in in vitro replication assays using
recombinant
29 templates lacking TP at one end (38). Full-length
29 ssDNA molecules may also be generated when the initiation rate is
reduced leading to only one initiation event/dsDNA molecule. However, as shown in Fig. 4, initiation rates of mutant TPs are similar to that
of wild-type TP (at short reaction times). Therefore, the finding of
full-length ssDNA molecules favors the model presented above in which
an origin containing one of the mutant TPs N80S, Y82L, or Y82S is inactive.
29 TP do not affect the function of primer protein but
alter function as parental TP. Thus, a mutant TP/DNA polymerase
heterodimer is able to recognize a wild-type origin allowing a first
round of DNA replication. However, once the mutant TP becomes parental TP the origin is inactive. This inactivation could be because of: (i)
the involvement of these parental TP residues in a "one side"
interaction with the primer TP/DNA polymerase heterodimer or (ii) a
reciprocal ("two side") involvement of these residues of the
parental-primer TP interactions. To distinguish between these two
possibilities, we carried out the following experiments. After
amplification for 40 min, when mutant primer TPs became mutant parental
TPs and DNA synthesis stops, new TP/DNA polymerase heterodimers were
added to the amplification reaction (Fig.
7). Upon the addition of wild-type TP/DNA
polymerase heterodimer, renewed incorporation of dAMP was observed. On
the contrary, addition of heterodimers with mutant TPs did not allow
dAMP incorporation. These results indicate that the origin containing a
mutant parental TP can be recognized (rescued) by a wild-type TP/DNA
polymerase heterodimer but not when it contains a mutant TP. Therefore,
the mutations introduced are critical when they are simultaneously present in both the primer and the parental TP, probably affecting their proper interaction required for a functional initiation reaction.
To test this possibility, an amplification assay was carried out for 40 min using mutant TP N80S to obtain DNA molecules containing mutant TP
at their origins. These products were then used as templates in
initiation reactions with either wild-type or mutant TP/DNA polymerase
heterodimers. Initiation reactions were only obtained with the
wild-type heterodimer (results not shown). The capacity of the
wild-type TP/DNA polymerase to rescue a mutant TP-DNA origin, both in
amplification and initiation assays, seems to rule out the possibility
that a mutant TP/DNA polymerase heterodimer forms an abortive
(nonproductive) complex that blocks the mutant TP-DNA origin.
Therefore, we consider that the inactivation of the origin is caused by
the lack of functional interactions between mutant primer and mutant
parental TP.
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Fig. 7.
In vitro 29 DNA
amplification with mutant TPs N80S or Y82L is recovered by addition of
wild-type TP/DNA polymerase heterodimer. First an amplification
assay was carried out in the presence of 10 ng of mutant TPs N80S or
Y82L for the indicated times at 30 °C (see "Materials and
Methods"). After 40 min, TP/DNA polymerase heterodimers containing
wild-type TP or mutant N80S or Y82L were added for a second
amplification assay, in which the previously amplified DNA is used as
template. The amount of incorporated [
-32P]dAMP was
measured at the indicated times.
29 TP did not alter its
primer function either in the presence or in the absence of parental TP
in the template, indicating that these residues are not involved in (i)
the interaction with
29 DNA polymerase and (ii) the formation of the
linkage to dAMP. Furthermore, these mutant TPs were not affected in the
transition step, a necessary step leading to TP/DNA polymerase
dissociation to enable processive DNA polymerization. However, once the
mutant primer TP became parental TP the replication origin was
inactivated to initiate additional replication rounds.
29 DNA replication origins occurs through direct interactions between primer and parental TP. In addition, we have provided evidence that residues Asn80 and Tyr82
of
29 TP are essential for such functional interactions at the origins. Moreover, for the first time we have found mutations that do
not affect the individual function of the TP either as primer or as
parental but are critical when present simultaneously in the primer and
parental TPs. These results support the existence of recognition
elements at the
29 DNA replication origin that imply complementary
interactions between primer and parental TP.
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ACKNOWLEDGEMENTS |
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We are very grateful to L. Villar for help during purification of proteins and to Dr. Maite Rejas for the electron microscopy work.
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FOOTNOTES |
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* This work was supported by research Grant 5RO1 GM27242-19 from the National Institutes of Health, by Grant PB93-0173 from the Dirección General de Investigación Científica y Técnica, by Grant ERBFMRX CT97 0125 from the European Economic Community, and by an institutional grant from Fundación Ramón Areces.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.
Recipient of a predoctoral fellowship from Ministerio de
Educación y Ciencia.
§ To whom correspondence should be addressed. Tel.: 3491-3978435; Fax: 3491-3978490; E-mail: msalas{at}cbm.uam.es.
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ABBREVIATIONS |
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The abbreviation used is: TP, terminal protein.
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REFERENCES |
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