DNA Polymerase in Thermococcus gorgonarius

Function

DNA polymerase has an important role in the development of life. DNA polymerase is an enzyme, which synthesizes new stands of DNA from preexisting DNA. It does this by adding one nucleotide at a time to the 3’ end of a growing strand. The new DNA strands are always synthesized from 5’ to the 3’ direction. (GENETICS BOOK) The original DNA stands are used as templates for the synthesis of new strands of DNA. DNA polymerases are the enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand. DNA polymerase I is composed of a single polypeptide that removes the RNA primers and replaces them with DNA. DNA polymerase III is responsible for the majority of the DNA replication and the proofreading from 3’ to 5’. ^[1] DNA follows Chargaff’s rule, which states that cytosine binds to guanine and adenine binds to thymine. ^[2] Each time a cell divides, DNA polymerase duplicates all of the cell’s DNA, and the cell passes one copy of DNA to each daughter cell. DNA polymerase roughly makes only one mistake every billion bases. Although Chargaff’s rule help makes the replication on DNA polymerase easier, an extra step of proofreading and cutting out the mismatched bases after replication is the reason why DNA polymerase is the most accurate enzyme. ^[1]

Structural Highlights

DNA polymerase from Thermococcus gorgonarius (Tgo) is composed of 2,319 nucleotides. Tgo a single polypeptide chain made up of 773 amino acids which are folded into five domains. The five domains in Tgo are the N-terminal, 3’ to 5’ exonuclease, palm, fingers, and the thumb. These five domain are arranged to form a ring shaped molecule. The structure of DNA polymerase can be described by using the “right hand model” which has a . The palm region (blue) catalyzes the phosphoric transfer. The finger region (green) interacts with the newly incoming nucleotides and the template bases that are paired together with the nucleotides. The thumb (red) helps position the DNA and with translocation. ^[3]

Stabilization

Thermococcus gorgonarius (Tgo) is a very thermophilic molecule with a growth range between 55-95 degrees Celsius. In order to replicate properly, the DNA polymerase must be stable and able to tolerate binding to the substrate DNA. Tgo polymerase contains 41% helix, 22% beta-stands, and 19% turns and also has an increase in hydrogen bonding.

There are two separate , which are formed at two different cysteine pairs in Thermococcus gorgonarius. Each sulfur atom is shown in yellow. Both of the cysteines are located on the palm domain. One disulfide bonds between cysteine (residue 428) and cysteine (442) stabilize the molecule at one of the folds between a helix in the palm domain and a helix located in the finger domain. While the other disulfide bond composed of cysteine (506) and cysteine (509) creates more stabilization for the helix in the palm domain at a helical turn. Sulfurs can withstand high temperature because their melting point is about 115 degrees Celsius. With these disulfide bond and structural folding this molecule is more thermostable.