Large-scale Molecular Dynamics Simulation of DNA Polymerase Beta
Complexed with Primer/Template DNA


    1. 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 enzyme.

    2. 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.

    3. 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.


    1. 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

    2. 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.

    3. 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.

    4. Dynamics of Some Key Hydrogen-bonds between Domains
      1. H-bond between Arg40 and Asp276 breaks at 1.3 ns as the polymerase begins to open;
      2. With thumb moving away from 8-kDa domain during the opening process, h-bond of Glu335/Ser44 breaks and that of Glu335/Lys48 forms;
      3. H-bond between Glu186 and Lys3 forms in the last 0.2 ns of the simulation;
      4. H-bond of Glu316/Arg182 remains throughout the simulation;
      5. Tyr271 is involved in three h-bonds with the incoming dNTP.

    5. 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