Structural Biochemistry/Nucleic Acid/DNA/Replication Process/DNA Polymerase

General information
The full process of DNA replication is comprised of the intricate and coordinated interplay of more than 20 proteins. In 1958, Arthur Kornberg and his colleagues separated DNA polymerase from E.Coli. DNA polymerase is the first known of the enzymes whose function is to promote the bond formation of the joining units that make up the DNA backbone. E.Coli has various numbers of DNA polymerases, assigned by Roman numerals, that play important roles in DNA replication and repair.

DNA polymerase is an enzyme. This enzyme synthesizes a new DNA strand from an old DNA template and also works to repair the DNA in order to avoid mutations. DNA polymerase catalyzes the formation of the phosphodiester bond which makes up the backbone of DNA molecules. It uses a magnesium ion in catalytic activity to balance the charge from the phosphate group.

Nucleotides are added to only the 3' end of the new strand; it is impossible for it to start a new chain on its own. Another DNA polymerase function is error correction - the correction of mistakes that were made in the new DNA strand. The entire DNA polymerase family consists of 7 different subgroups: A, B, C, D, X, Y and RT. Eukaryotes have at least 15 different DNA polymerases. However, none of the eukaryotic polymerases can remove primers, and only the elongation polymerases can proofread the sequence.

Although there are different types of DNA polymerases, all have common structural features. Additionally, even though DNA polymerases differ greatly in detail, they have very similar overall shape. There are at least 5 structural classes of DNA polymerase that have been identified. They take the shape of a hand with specific regions referred to as the fingers, the palm, and the thumb. In all classes of DNA polymerase, the thumb and finger wraps the DNA, holding it across the active site of the enzyme, while the palm releases residues that comprise this active site. Moreover, all DNA polymerases use similar strategies in the catalyzation of the reaction.



General Formulation
DNA polymerases are the catalysts in the step-by-step addition of deoxyribonucleotide units to a DNA chain. The reaction catalyzed is

(DNA)n + dNTP ↔ (DNA)n+1 + PPi

where dNTP stands for any deoxyribonucleotide and PPi is a pyrophosphate ion.

Requirements
1. All four activated precursors are needed for the reaction to occur, the deoxynucleotide 5’-triphosphate dATP, dGTP, dCTP, and dTTP, in addition to Mg2+ ions. Typically, two of the metal ions will take part in the reaction. One will interact with the primer while the other with dNTP. The carboxylate groups of the residues in dNTP bind the two metal ions in place.

2. The new DNA chain is constructed directly on a pre-existing DNA template. DNA polymerases can only work efficiently as a catalyst in the formation of phosphodiester bonds if the base on the incoming nucleotide triphosphate is complementary to that of the template strand. In other words, DNA polymerase is an enzyme that synthesizes a product by interpreting the existing DNA strand as a template and produces the complementary sequence of the template into a new strand.

3. DNA polymerases necessitate the presence of a primer to start synthesis. The reaction catalyzed by DNA polymerases that works to elongate the chain is a nucleophilic attack by the 3’OH terminus of the growing chain on the innermost phosphorus atom of the deoxynucleotide triphosphate. Therefore, a primer strand with a free 3'-OH group must be bound to the template strand from the start. This primer is formed from RNA synthesis. Due to the fact that RNA can form without a primer, it starts the synthesis of DNA. Once the complementary DNA is formed and the synthesis has been initiated, the RNA piece will be removed and then replaced by the proper DNA sequence. A phosphodiester bridge is formed from the reaction and pyrophosphate is released. The ensuing hydrolysis of pyrophosphate that results in the creation of two ions of orthophosphate (Pi) by pyrophosphate assists to drive the polymerization forward. This elongation process of the DNA chain proceeds in the 5’-to- 3’ direction.

4. Many DNA polymerases are able to remove the mismatched nucleotides as a method of mistake correction in DNA. The polymerases possess a distinct nuclease activity that allows them to eliminate incorrect bases through a separate reaction. DNA polymerase will reverse its direction by one base pair and excise the incorrect base to replace it with the proper one and continue with the rest of replication. Due to this 3' to 5' exonuclease activity, DNA replication has a remarkably high dependability. This step process is also called proofreading. However, it is not completely perfect, which is why natural mutations and related diseases can still arise.

Eukaryotic DNA Polymerases
DNA Polymerases play a key role in the synthesis of DNA. Without these players, life would cease to exist. These polymerases are multi-subunit complexes that function very uniquely. It requires different components to work together to function efficiently. Polymerases act upon single-stranded strands (specifically to the template), to synthesize a strand that is complementary. In eukaryotic cells, there are 5 families of DNA polymerase. These can encode into different (up to as many as 15) enzymes. Critical for DNA replication are three DNA polymerases: Polymerase α-primase, Polymerase δ, and Polymerase ε. These three polymerases function at the replication fork of the DNA strands. The DNA strands are unwounded by MCM helicase, which is part of a CMG complex (Cdc45-MCM-GINS). It is Polymerase α- primase that initiates replication on the leading and lagging strand. It is here that the RNA primers (about 10 nucleotides) are laid down.

After the initiation, Polymerase δ and ε are brought to the complex and tethered. They function to increase the productivity of the different enzymes. Specifically, Pol δ synthesizes on the lagging strand while the Pol ε synthesizes on the leading strand. The roles of these polymerases were found by genetic experiments. For Pol ε, a mutation was placed on the active site. This increased the rate of enzymes activity, and leave behind a signature in the regions of activity. With the involvement of reporter genes, it proved that the Pol ε did indeed participate in the synthesis of the leading strand. The same genetics were done for Pol δ to prove its activity with the lagging strand.

A consistent correctness is necessary with the implementation of the bases. An incorporation fortunately occurs only every 10,000 replicated base pairs. But when it does occur in the DNA primer strand, it must be moved out from the polymerase and to the exonuclease domain. It is there that it is proofread and allow for continuation of a stable domain. [1]

Polymerase Families
Central to life, polymerases have been put under study in search of its structure as well as roles. To date, there have been 7 different families (or domains). There are 5 unique to eukaryotic cells. More families are unique to bacteria and archaea. In these polymerase families, there is a core structure: palm, finger, and thumb domains. From there the families diverge to their specific cellular functions. The 7 families are labeled with letters: A, B, C, D, X, Y, and reverse transcriptase. Family A includes Pol I polymerase, which functions to repair nucleotides. It also includes Okazaki fragments, which takes part in the replication of the lagging strand. Family B includes the eukaryotic polymerase sigma, alpha, as well as epsilon. Family C harbors the Polymerase III, which XXX. Family D includes polymerases that are exclusive to archaea. Family X as well as Y include enzymes that do repairing.



Within the Eukaryotic DNA Polymerase Structures
As it was earlier noted that the polymerases are multi-unit entities, it holds true that they are very complex. The structures are comprised of a large catalytic subunit (part of the B family), and then many other smaller subunits. The architecture of the B family polymerases are consistent: a N-terminal domain, 3’-5’ exonuclease domain, palm, finger, and thumb domains; in a ring-like structure. The catalytic subunit of all the eukaryotic polymerases are assumed to be related and come from a common ancestor via gene duplication. But studies do show that the catalytic subunit of the ε is larger than the other two due to additional sequences.

Obtaining structures that are in high resolution is essential for further analysis of polymerases. To date, there has been a lot of progress in formulating the structures of the different subunits that make up the polymerases, but only at low-resolutions. The first structure reported was the cryo-EM structure of the Pol ε. Researchers aim to work towards high resolution structures because it came allow further understanding of the fidelity of DNA synthesis, and the highly regulated genome that is maintained in all of the eukaryotic cells. Furthermore, it would allow design of genetic experiments to explore the interactions of and within the complexes.[2]