Large-scale Molecular Dynamics Simulation of DNA Polymerase Beta
Complexed with Primer/Template DNA
- Biological Background
DNA repair mechanisms are vital to life, since DNA is frequently damaged by a
variety of chemical and physical agents. Most of the damaged DNA bases are excised by
one of two major pathways in both prokaryotic and eukaryotic cells: base excision repair
(BER) and nucleotide excision repair (NER). While the NER pathway generally repairs
bulky adducts, e.g., UV-induced dimers and adducts of benzo[a]pyrene
metabolites, the BER pathway repairs smaller base modifications and is limited to
relatively short excision gap created by deamination, oxidation,
alkylation, and ultraviolet radiation. In the BER pathway, the damaged base
is identified and then removed by DNA glycosylases, resulting in
an AP site. This AP site is then recognized and cleaved
by AP endonucleases, and the remaining deoxyribose
phosphate (dRP) residue is excised by a phosphodiesterase
or lyase. A DNA polymerase then fills in the small gap
in the DNA by selecting the correct 2'-deoxyribonucleoside 5'-triphosphate (dNTP)
complementary to the template for reaction with the
3'-end of the primer strand with release of pyrophosphate (PPi),
and finally the nicked phosphodiester backbone is sealed by a DNA ligase
- Architecture of Polymerase beta
Polymerase beta consists of two domains (335 residues): the 8-kDa N-terminal domain
and the 31-kDa C-terminal domain. The latter is like a hand with thumb, fingers, and
palm subdomain, which is a commom architectural feature shared by all polymerases.
The palm's role is catalysis of the phosphoryl transfer
reaction, and the thumb subdomain controls interactions with the
incoming nucleoside triphosphate and the corresponding
template base. The fingers may play a key role in positioning
the duplex DNA into the wide U-shaped cleft of the enzyme
and in processivity and translocation.
- Crystal Pol-beta/DNA complexes in Closed and Open States
Three crystal complexes of human pol-beta have been obtained: the binary complex
with a DNA substrate containing a one nucleotide gap
(Pol-beta.Gap), the ternary complex which contains an
incoming ddCTP (Pol-beta.Gap.ddCTP) ,
and the binary product complex containing only nicked DNA
(Pol-beta.Nick) . The ternary complex is in the
``closed'' state and both binary complexes are in
the ``open'' state. The difference between
those two states is the large movement of thumb subdomain, as well as position
changes for some characteristic residues, as shown in the following figure.
- Sequential Motions During the Opening Process of Polymerase Beta
Our molecular dynamics simulations suggest the following series of sequential related
motions for the full opening process, involving the key amino residues
Phe272 and Arg258. First, the aromatic ring of Phe272 flips away from Asp192; second,
the pol-beta opens through a large thumb movement; third, the hydrogen bonds of Arg258
with Glu295 and with Tyr296 are broken, and Arg258 rotates toward Asp192.
| Step 1: Phe272 Flips
|| Step 2: Thumb Movement
|| Step 3: Arg258 Rotation
- Water Density Changes in the Active Site
The water density near the new base pair increases in the polymerase
opening process, as seen the following figure. The low water density
around the active site in the closed complex results from the new
base pair being tightly sandwiched by the alpha-helix~N and its neighbor
base pair. With thumb opening, alpha-helix~N moves away from the new
base pair and more water molecules come close to the active site.
- Catalytic Mg2+ ion Departure May Be Slow in the Polymerase Opening
One sodium ion was observed to coordinate with three aspartates in the active site
during the last 1.2 ns simulation. These residues, Asp190, Asp192, ans Asp256,
are normally bound to the catalytic Mg2+ in the close form. Thus, this Na+ may be the
surrogate for the catalytic Mg2+, which remains in the active site throughout the
simulation. The binding or release of the catalytic Mg2+, as well as the rearrangement
of Arg258 may be the slow step before and after the chemical DNA incorporation.
- Dynamics of Some Key Hydrogen-bonds between Domains
- H-bond between Arg40 and Asp276 breaks at 1.3 ns as the polymerase begins to open;
- With thumb moving away from 8-kDa domain during the opening process,
h-bond of Glu335/Ser44 breaks and that of Glu335/Lys48 forms;
- H-bond between Glu186 and Lys3 forms in the last 0.2 ns of the simulation;
- H-bond of Glu316/Arg182 remains throughout the simulation;
- Tyr271 is involved in three h-bonds with the incoming dNTP.
- A-like DNA in the Active Site
DNA is A-like near the polymerase active site in a number
of crystal structures containing primer/template complexes.
Analysis of key parameters from our simulation reveals
such trends for the DNA in the active site.
The widened minor groove is biologically significant in that
it permits the primer/template DNA to better contact the active
site residues such as Arg283, and Tyr271,
thereby assisting in positioning the 3'-terminus of the
primer for the nucleotidyl transfer reaction.
Please see the detailed description in the manuscript.
Related Works and DNA polymerase beta movies in S. Wilson's Group