From the
Bacteriophage 29 DNA polymerase, the product of the viral
gene 2, was originally characterized as a protein involved in the
initiation of
29 DNA replication based on both in vivo(1) and in vitro(2, 3, 4) studies. The cloning of gene
2(5) , the overproduction and purification of its
product(6) , and the development of an in vitro system
for complete
29 DNA replication (7) allowed the
characterization of protein p2 as the viral DNA replicase(8) .
This monomeric enzyme, with a molecular mass of only about 66 kDa,
catalyzes two distinguishable synthetic reactions: 1) DNA
polymerization, as any other DNA-dependent DNA polymerase, with
insertion discrimination values ranging from 10
to 10
and with an efficiency of mismatch elongation
10
-10
-fold lower than that of a properly
paired primer terminus(9) ; 2) terminal protein (TP) (
)deoxynucleotidylation, which consists of the formation of
a covalent linkage (phosphoester) between the hydroxyl group of a
specific serine residue (Ser
) in
29 TP and 5`-dAMP,
requires the presence of divalent metal ions and is strongly stimulated
by the presence of the viral DNA replication origins. By means of this
reaction, in which the TP is acting as a primer,
29 DNA
polymerase catalyzes the initiation step of
29 DNA
replication(5, 8) . In addition to the synthetic
activities,
29 DNA polymerase has two degradative activities: 1)
pyrophosphorolysis, the polymerization reversal, whose physiological
significance is still unclear(10) ; 2) 3`-5`-exonuclease,
shown to be involved in a proofreading
function(11, 12) . This activity, kinetically
characterized using ssDNA as substrate and Mg
as
metal activator(13) , degrades processively DNA substrates
longer than six nucleotides, the catalytic constant being 500
s
. When the DNA length is reduced below 6-4
nucleotides, the
29 DNA polymerase-ssDNA complex dissociates at a
rate of 1 s
.
The multiple enzymatic activities of
29 DNA polymerase (summarized in Table 1) allow this enzyme
to be the only polymerase involved in the replication of the
29
genome(7, 14) . Moreover, the enzyme has two intrinsic
properties: high processivity (>70 kilobases) and strand
displacement ability(15) . Based on this enzymatic potential,
complete replication of both DNA strands can proceed continuously from
each terminal priming event, without the need of synthesis of
RNA-primed Okazaki fragments and making unnecessary the participation
of accessory proteins and DNA helicases. The efficiency of the
protein-primed initiation reaction is in part guaranteed by the
previous formation of a heterodimer between TP and DNA
polymerase(16) , whereas the nucleotide specificity, as in
normal DNA polymerization, is dictated by the DNA
template(17) .
A ``Sliding-back'' Mechanism to Initiate TP-primed
DNA Replication
It has been shown that 29 DNA polymerase does not start
replication at the first base of the genome but employs the second
position from the 3`-end of the template for the initial base pairing
and formation of the corresponding TP-dAMP complex at each DNA end. The
DNA ends (telomeres) are recovered by a specific mechanism, so called
``sliding-back,'' that is based on a 3`-terminal repetition
of two T residues. This reiteration permits, prior to DNA elongation,
the asymmetric translocation of the initiation product, TP-dAMP, to be
paired with the first T residue(18) . The fact that
TP-containing genomes, either from virus or linear
plasmids(7) , contain some kind of sequence repetitions at
their ends supports the hypothesis that the ``sliding-back''
mechanism could be a common feature of protein-primed replication
systems(18) . This proposal has been demonstrated in the case
of bacteriophages PRD1 from Escherichia coli and Cp1 from Streptococcus pneumoniae and in adenovirus. PRD1 DNA
polymerase initiates replication at the fourth nucleotide of the
terminal 3`-CCCC repetition (19) and Cp1 DNA polymerase at the
third nucleotide of the terminal 3`-TTT repetition, (
)the
end being recovered in both cases by a ``stepwise
sliding-back'' mechanism. Adenovirus DNA polymerase initiates
replication at the fourth nucleotide of the terminal repetition
3`-GTAGTA, followed by a preelongation step that originates TP-CAT, the
end being recovered by a ``jumping-back'' step (20) .
Structural Mapping of the Enzymatic Activities of
29 DNA Polymerase
Figure 1:
Structure-function
studies of 29 DNA polymerase. A, relative arrangement of
the most conserved regions among prokaryotic and eukaryotic DNA
polymerases. The amino acid sequence of
29 DNA polymerase (572
amino acids) is represented by a bar, with the N terminus at
the left. Gray and filled-in regions indicate the
predicted 3`-5`-exonuclease and DNA polymerization domains,
respectively. The area in between these two domains (ct) has
been involved in the communication or cross-talk among these two
domains. Alternative nomenclature for the regions (boxed) that
contain the motifs (44) is indicated. A, B,
and C correspond to motifs that are generally conserved among
different classes of nucleic acid-synthesizing enzymes(27) . B, proposed role for individual residues forming highly
conserved N-terminal and C-terminal motifs of
29 DNA polymerase,
as defined by site-directed mutagenesis. Motifs are represented in
single-letter notation, where x indicates any amino acid.
Alternative residues for a particular position are separated by a bar. A summary of the mutational analysis carried out in
29 DNA polymerase is described in the
text.
It has been described that the binding of
29 DNA polymerase to DNA primer-template structures is largely
enhanced by the presence of metal ions known to activate DNA
polymerization(30) . This behavior suggests that metal-assisted
DNA binding could also increase the efficiency of DNA translocation,
thus favoring the processivity of
29 DNA polymerase.
Communication between the N-terminal
and C-terminal Domains: Coordination between Synthesis and Degradation
As described before, the mutational analysis carried out
along the 29 DNA polymerase molecule allowed demonstration of the
existence of two structurally independent domains containing the
synthetic and degradative activities of this enzyme. However, for an
effective proofreading of DNA polymerization errors, a mechanism for
coordinating DNA polymerization and DNA excision must exist, relying on
a structural and functional communication or cross-talk between the
N-terminal and C-terminal domains. The basis of this communication,
specially important in the case of processive DNA polymerases, involves
the intramolecular switching of the primer terminus between the
polymerization and 3`-5`-exonuclease active sites.
By
site-directed mutagenesis of 29 DNA polymerase, it has been
recently demonstrated that the conserved motif
``YXG(G/A),'' located between the
3`-5`-exonuclease and polymerization domains of eukaryotic type
DNA polymerases (Fig. 1), is a DNA binding motif that plays a
role in the coordination between DNA synthesis and proofreading. (
)We propose that residues Tyr
and Phe
of
29 DNA polymerase are primarily involved in the
stabilization of template-primer structures at the polymerization
active site, playing also a role in the movement (switching) of the
primer terminus between the polymerase and exonuclease active sites.
This dual role could be achieved if the ``YXG(G/A)''
motif is involved in a conformational change, triggered by the
unstabilization produced by insertion of a mismatched nucleotide. In
addition to this motif, other amino acid residues of
29 DNA
polymerase have been implicated in stabilization of the primer terminus
at both polymerization (Thr
, Arg
,
Lys
, and Tyr
) and exonuclease (Thr
and Asn
) active sites, playing in this case an
indirect role in the dynamics of DNA interaction required to coordinate
polymerization and proofreading.
In addition to the structure-function studies that are
extrapolatable to most DNA-dependent DNA polymerases, one of the main
goals of our research is the characterization of the structural basis
for the intrinsic high processivity of 29 DNA polymerase. To
approach this problem, we will search for specific subdomains that
could lead to a proliferating cell nuclear antigen-like topological
interaction with DNA. As it has been described for both proliferating
cell nuclear antigen (45) and the
subunit of E. coli DNA polymerase III(46) , such a strong interaction would
be dissociated only when reaching a DNA end, as should be the case
after completing replication of the linear
29 DNA molecule. Of
additional interest is understanding how
29 DNA polymerase is
able to use both a protein and DNA as primers, and the dynamics of
interactions occurring at the transition between TP-primed initiation
and the elongation stage of
29 DNA replication. One of the most
intriguing questions is whether, after formation of the TP-dAMP
initiation complex, TP and
29 DNA polymerase must dissociate
either as a consequence of the special translocation step
(``sliding-back''), necessary to accommodate the newly
created primer terminus in an adequate position to accept the next
incoming dNTP, or after the synthesis of a short DNA suitable to be
used as primer.
Attempts to obtain 29 DNA polymerase crystals
adequate for x-ray diffraction analysis were not successful so far.
Other approaches, such as the crystallization of
29 DNA
polymerase complexed with TP, will be also explored to elucidate the
structural basis for the vast potential of this monomeric enzyme.