The Bacteriophage T7
DNA Replication Complex
Michael E. Ward(1)
and David Marcey(2)
© David Marcey, 2001
II. Structural Features
III. DNA Synthesis
IV. T7 Polymerase Interactions with the Primer-Template
Note: This exhibit is best viewed if the cue buttons ( ) are pressed in sequence and if the viewer does not independently manipulate the molecule on the left.
details of DNA synthesis in higher organisms is not thoroughly understood, in
part because so many molecules are involved in the process. The bacteriophage
T7 DNA replication complex is a good model system for the study of the mechanism
of DNA synthesis because it consists of relatively few proteins. Elucidating
the mechanism of T7 DNA replication will likely provide insights into the workings
of more complex DNA replication machines. This exhibit, based on the recent
work of Doublie, et al. (1998), explores
the intricacies of DNA synthesis in the phage T7 DNA replication complex. The
exhibit focuses on how processivity is maintained during DNA synthesis, on how
metal cations are involved in nucleotide addition, and on how T7 DNA polymerase
recognizes specific nucleotides.
the left is the crystal
structure of T7 DNA polymerase and a short stretch of dsDNA (primer
strands). The polymerase
is caught in the act of adding a nucleotide to
the 3' end of the newly synthesized DNA primer . There are several proteins involved in T7 viral DNA synthesis:
1) A hexameric T7 primase-helicase that unwinds and primes the DNA (not shown);
2) A T7 single-stranded DNA binding protein that binds unwound, ssDNA in anticipation
of DNA synthesis (not shown); 3) An 80K T7 DNA polymerase , and; 4) E. Coli thioredoxin
, a bound processivity factor that prevents the polymerase
from falling off the DNA template.
The polymerase structure can be thought of as an
open right hand, composed of a thumb domain
that binds to thioredoxin, a
in which catalytic activity resides, a
palm domain that cradles the DNA, and a terminal exonuclease
The polymerase domains are built mostly of alpha helices, which play important
roles in nucleotide recognition as well as overall protein structure . Several domains also contain b- sheets.
Shown here is a close up of the DNA primer strand (the DNA strand that is being extended) and template strand (the parental strand). Also shown is an incoming nucleotide that is being added to the growing primer strand. Note the hydrogen bonding between complementary base pairs (denoted by dashed lines). Attached to the 3' end of the primer strand is a dideoxynucleotide, lacking a 3' OH. Click here to add a 3'OH to this nucleotide (the hydrogen is not shown). During DNA primer extension, the oxygen of this 3'OH group attacks the alpha phosphorous atom of the incoming nucleotide's 5' phosphate group in an SN2 reaction. This results in the loss of two phosphates from the incoming nucleotide. There are two magnesium ions , one juxtaposed to the 3' OH group and one in close proximity to the phosphorous atom, that facilitate this reaction. The magnesium ion next to the 3' OH stabilizes the ionized form of oxygen (O-), increasing its nucleophilicity and leading to the SN2 attack on the alpha phosphate. The second magnesium ion contributes to the reaction by stabilizing negative charges on the diphosphate leaving group.
explore how these magnesium ions are positioned by T7 DNA polymerase.
Here is a portion of the palm domain involved in
this positioning . The oxygens of the side chains of Asp 475 and Asp654,
as well as the main chain oxygen of Ala476, are coordinated (via
electrostatic attraction) with the magnesium ion
that is associated with the incoming nucleotide. The
is coordinated with other oxygens
on Asp475 and Asp654, as well as with H20
not shown) associated with the finger domain of the protein .
There are multiple interactions between the incoming nucleotide and T7 DNA polymerase. These interactions serve to stabilize the incoming nucleotide and to increase the fidelity of the polymerase. It is theorized that different, and unique, interactions occur for each type of incoming nucleotide (A, T, C, or G) and the corresponding template nucleotide such that A can only bind with T, and G can only bind with C. In the crystal structure at left, C is the template nucleotide, and G is the incoming nucleotide.
A number of polymerase amino acid side chains bind the cytosine template. Two residues of the finger domain bind to the phosphates that flank this cytosine: His607 and Gly533 (nitrogens on the protein are electrostatically attracted to oxygens on the phosphate groups of the DNA template) . In addition, the a- carbon of Gly527 (flashing) stacks on top of the of the cytosine pyrimidine ring . This stacking is especially important as it determines which nucleotide will be allowed to enter the active site of the enzyme (see below).
Next, let's focus on the interactions of protein side chains and the incoming nucleotide. Glu480 and Tyr526, both residues of the finger domain, bind to the sugar ring (flashing) of the incoming dGTP via van der Wall's forces . Also, there are direct interactions between the diphosphate leaving group and finger domain residues (Lys522, His506, Arg518, and Tyr526) . The interactions are primarily through the electrostatic attraction of side chain nitrogens to nucleotide oxygens. These residues form a "glove" around the incoming nucleotide, precisely positioning it in anticipation of the subsequent 3' OH nucleophilic attack. Note that Lys522, Arg518, and Tyr526 are all on the same alpha helix as Gly527; this arrangement dictates the distance between the incoming nucleotide and the template nucleotide, prohibiting the formation of pyrimidine-pyrimidine or purine-purine binding.
T7 DNA polymerase can differentiate between purines and pyrimidines--but how does the enzyme make sure that the correct purine-pyrimidine nucleotide pair forms (i.e., G-C and not A-C)? It appears that mismatched base pairing (between A-C, for example) would disrupt hydrogen bonding between the nucleotide pair directly below the incoming nucleotide and residues Arg429 (palm) and Gln615 (finger) . Also, mismatching would disrupt interactions between the incoming nucleotide and Arg429 .
The ability of an organism to replicate its genome efficiently and accurately is paramount to its survival. It seems that many of the mechanisms T7 DNA polymerase uses to catalyze DNA replication, such as the two metal-ion catalysis mechanism, have been broadly conserved in evolution. It is now becoming clear that DNA polymerases can accurately select the correct incoming nucleotide before its incorporation into the extending DNA molecule, thus increasing the accuracy of DNA replication.
Doublie S., Tabor S., Long
A., Richardson C., and Ellenberger T. (1998). Crystal Structure
of a Bacteriophage T7 DNA Replication complex at 2.2 A Resolution. Nature
Kenyon College, Gambier, Ohio. A first draft of this exhibit was created for
D. Marcey's Molecular Biology class, Biology 63.
2, Kenyon College, Gambier, Ohio. Present address: California Lutheran University. Address correspondence to this author (see below).