Bacteriophage
T7 DNA Polymerase
Michael Ward, Jennifer Lovick, and David Marcey
© 2006
I.
Introduction
II. Structural Features
III. DNA Synthesis
IV. References
Directions
If
prompted, allow your browser to view blocked content. The OMM
now uses the Signed Jmol Applet. No files from this site can
damage your computer. Just check the appropriate box and then
click run.
This exhibit displays molecules in the left part of the screen,
and text that addresses structure-function relationships of
the molecules in the right part (below). Use the scrollbar to
the right to scroll through the text of this exhibit.
To evoke renderings of the molecule that illustrate particular
points, click the radio buttons:
Please
click the load PDB buttons,
, when present.
To
reset the molecule, use the reset buttons:
If
you resize your browser window, simply refresh the page in order
to restore proper viewing. |
I.
Introduction
The
mechanistic details of DNA synthesis in higher organisms is not completely
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.
At left is
the crystal structure of a T7 DNA polymerase complex and a short stretch
of double stranded DNA with the primer
and
template
strands
indicated. The polymerase
is caught in the process of adding a
nucleotide to the
3' end of the primer
DNA strand.
return
to beginning
II. Structural
Features
The
crystal structure of the polymerase complex reveals two proteins essential
to replication of the phage genome:
- the
polymerase proper,
an 80kD phage
protein with both polymerase and exonuclease
catalytic activities;
-
thioredoxin,
a host-encoded (E.
coli) processivity factor that
anchors the polymerase
to the DNA template for extended periods of DNA synthesis.
Like
many other DNA polymerases, the
T7 polymerase can
be visualized as an open right hand, composed of a thumb
domain that
binds to thioredoxin,
a fingers
domain in
which catalytic activity resides, and
a palm
domain.
The
DNA is cradled in this open hand, with the palm
domain forming a plate at the bottom of the cleft formed by
the thumb and fingers
domains.
An N-terminal exonuclease
domain
abuts the palm
domain.
The
polymerase
domains are built mostly of alpha-helices,
which play important roles in nucleotide recognition as well as in
maintaining overall 3-D structure. Several domains also include beta-sheets.
For example, the palm domain contains a prominent beta-sheet
that forms the bottom of the DNA-binding cleft.
There
are several other key proteins involved in T7 viral DNA synthesis
that are not shown in the structure at left: 1) a hexameric T7 primase-helicase
that unwinds and primes the DNA; 2) a T7 single-stranded DNA
binding protein that binds unwound, ssDNA in anticipation of DNA synthesis.
return
to beginning
III. DNA Synthesis
Shown at left is a close
up of the DNA primer strand (the daughter
DNA strand that is being built by polymerization of nucleotides) and
the template
strand (the parental strand).
Also shown is an incoming nucleotide
(dGTP) that is being added to the growing
primer
strand. The
last added nucleotide (dA) is indicated
in yellow. Hydrogen bonding between complementary
base pairs is indicated by dashed lines.
Note the free 3'OH of the last added nucleotide on the primer strand (H not shown). During DNA primer extension, this hydroxyl oxygen attacks the alpha phosphorous atom of the incoming nucleotide in an SN2 reaction.
This attack links the incoming nucleotide to the primer strand, resulting in the loss of two phosphates from the incoming nucleotide.
A universal feature of nucleic acid polymerases is the use of two metal cations to catalyze the addition of new nucleotides to a growing chain. In the T7 DNA polymerase active site, two magnesium ions are positioned to facilitate the polymerization reaction. One Mg++ is juxtaposed to the 3' OH of the last added nucleotide on the primer strand. This stabilizes the ionized form of oxygen (O-), increasing its nucleophilicity, which leads to the SN2 attack on the alpha phosphorous atom. The other Mg++ contributes to the reaction by stabilizing negative charges on the diphosphate leaving group.
Now let's explore how these magnesium ions are positioned by T7 DNA polymerase.
Three catalytic site residues of the palm domain (Asp475, Asp654, and Ala476) as well as several water molecules (H's not shown) associated with the finger domain coordinate the two Mg++ atoms in the heart of the DNA binding cleft of the polymerase.
return
to beginning
IV. References
Doublie S., Tabor
S., Long A., Richardson C., and Ellenberger T.: Crystal Structure
of a Bacteriophage T7 DNA Replication complex at 2.2 A Resolution.
Nature 391: 251-258 (1998).
return
to beginning
|