DNA Polymerase Eta
Created by: Matt Lycas
The study of DNA polymerase has allowed for a much greater understanding of the process of DNA replication. It facilitates the copying of a DNA strand from a daughter cell, and translocates it across the DNA strand with a high degree of accuracy. DNA polymerase eta in complex with undamaged DNA is a eukaryotic DNA polymerase, and it is in the family of DNA polymerase Y. Knockout of this enzyme in yeast cells yields a high susceptibility to ultraviolet light damage (Biertümpfel, C., 2010). Removing the trait in people can result in skin cancers. These polymerases are notable for their unique structure, their ability to repair damaged DNA, and low fidelity. DNA polymerase eta (PDB "3MFH") has the specific ability of repairing damage from ultraviolet radiation. The molecular weight of this protein is 58735.54 and the isoelectric point is 7.17 (Expasy, 2012).
This protein functions by forming a structure with a DNA molecule and the nucleotide being added. The primary structutre is responsible for the globular shape and function of the protein. There are many polar residures on the outside of the protein, and nonpolar residues faceing inside. As an enzyme that fascilitates a motion intensive process, this nonpolar/polar relationship is not absolute. At any given stage the sections will have different contact with the surrounding system. The quaternary structure is formed from the amino acid chain of the protein and the DNA that it is polymerizing. The protein forms a complex joining the incoming nucleotide with the DNA strand. This enzyme is typically characterized by a similar tertiary architecture: three structures consisting of the finger, palm, and thumb (Rothwell, P. J., 2005). Polymerases in this family have an additional region. It is cited in the literature as the little finger (LF), wrist, or polymerase associated domain (PAD). Most interfamily differences arise in the finger and thumb structures. DNA polymerases in the Y family bind with the DNA in a way allowing for a greater solvent contact with the DNA molecule. It is 600 Å from the molecular surface, as opposed to 1000 Å as seen with DNA polymerase A and B (Rothwell, P. J., 2005). DNA polymerases in the Y family are able to keep hold on the DNA with the extra PAD structure. The PAD is composed of two alpha helices and four beta strands. These are made from residues 395-509. It is the 4 beta sheets that allow for this decrease in DNA penetration into the protein. These bind onto the DNA polymerase in addition to the thumb. The little finger has an affinity for the major groove of the DNA molecule. The binding between the DNA molecule and the protein is mediated through weak protein interactions. Hydrogen bonds form between the two, connecting the DNA chain to the beta strands B12 and B14. Important residues for this binding include Ser-394, Met-396, Asn-398, Asn-400, and Ser-458. These residues reside in beta sheets. The hydrogen bonds between the DNA and the little finger take place more on the template strand (T) opposed to the primer strand (P). They form on T4, T3, T2, T1, and P7. (Silversterin, T. D., 2011) The DNA is bound on the other sides by the thumb region. The thumb makes contact with the minor groove of the DNA molecule. The decreased overall contact with the nucleotide results in the ternary complex’s lower fidelity opposed to that of other DNA polymerases. The finger region is smaller and lacks the components found in other polymerases to bind to the DNA. This allows for it to opperate over larger obstructions in the DNA sequence, but also results in codeing with lower fidelity.
This DNA polymerase is noted for its lower fidelity. This is due to the free nucleotide allowed to have greater exposure to solvents. The nucleotide binds to the enzyme by positioning in the fingers and the palm structures. Tyr-64 and Arg-67 from the finger structure and Lys-279 from the palm make hydrogen bonds to stabilize the binding of the nucleotide. Phe-35 interacts with the sugar in the dATP. Studies of the kinetics reveal that the acquisition of anucleotide is a two step process. The enzyme is 5.4 times more likely to perform the correct matching of nucleotides than the incorrect matching (Washington, M. T.). Comparisons of the equilibrium constant K for the binding of the nucleotide between the binding of correct base pairs and of incorrect base pairs shows that the binding is most likely not hindered by geometric constraints from the enzyme. The change in ΔG is minimal compared to that for other polymerases (Washington, M. T.). The protein first binds to the DNA molecule, then incorporates the nucleotide. The protein undergoes a conformational change, allowing a phosphodiester bond to form between the nucleotide and the DNA. The enzyme returns to its original conformation and either translocates along the DNA or releases it. This protein conformational change is what discriminates for the correct nucleotide. This makes it an induced fit mechanism to function (Fiala. K., 2004).
The UmuC domain is what accounts for the UV light mutagenesis repair. It is also found, according to the expasy databases, in bacteria umuC protein, E. Coli mucB protein, Salmonella Typhimurium impB protein, Salmonella Typhimurium samB, Bacterial DNA polymerase IV, and yeast REV1 protein (UmuC, 2012). This is the sequence Ilu-29 to Gly-313. This site contains two ligands and an active site. Residues Glu-39 and Glu-156 both bind to magnesium ions. The pH, in vivo, will deprotonate the carboxylic acid, allowing electrophilic oxygen to bind strongly to the positively charged magnesium ions. The active site is at residue 162-Glu. This is located within the palm region of the polymerase. There are no important disulfide bridges in this domain.
With the growing number of known protein structures, the Dali server provides a comparison of the target protein to others in terms of 3D structure. DNA polymerase was found to be most similar to DNA polymerase Iota (PDB "3H40"), DNA polymerase IV (PDB "2BQR"), DNA polymerase Kappa (PDB "1T94"), and DNA repair protein REV1 (PDB "3GQC"). They had Z scores of 31.5, 27.5, 21.3, and 4.6 respectively (Dali query, 2012). These are all classified as DNA polymerase Y proteins, and the similarity in tertiary structure matches the similarity in function. DNA polymerase IV is in prokaryotic systems and the others are in eukaryotic system. These proteins in general had conserved primary structures between residues approximately 37-165 and 240-505. These ranges include sections from each structure, but contain completely the fingers, thumb, and little finger structures. The palm area is what is most varied between this protein and the others in its family.
Analysis through the BLAST sequence analysis tool shows relations in primary structure between proteins. Proteins in the DNA Y family were again most similar in this respect (Conserved domains, 2012). DNA polymerase kappa has an E score of 5.98*10^-15, iota at 5.60 * 10^-11, and REV1at 3.66 * 10^-7 (BLAST, 2012). The E value approaches 0 as the two proteins become more similar, in comparison with a database of all other known primary structures. The score closest to zero will reflect that the protein sequence is similar and that it is long enough to be uniquely found between these two proteins.
Ultraviolet light causes thymine-thymine dimers to form in DNA (Goodsell, D.S., 2001). The high energy photons break apart the pyrimidine’s bond. If there is another broken pyrimidine in the sequence, the neighboring bases will bond, forming a cyclobutane. This happens 50 to 100 times a second to the DNA in each skin cell, making the role of this polymerase essential (Goodsell, D.S., 2001). The cyclopyrimidine dimer is sterically bulkier than normal DNA. DNA polymerase eta has a larger active site to account for this. The residues that do bind with the DNA reinforce the stable B-form conformation of DNA. Lastly, this cyclopyrimidine dimer is stabilized with nonpolar residues located near its position to decrease the hydrophobic effect (Ling, H., 2001).
DNA polymerase Iota (PDB "3H40") is most similar to the target protein by Z score and also has an E value of 5.6 * 10^-11, very close to zero. This enzyme has been hypothesized to have evolved from DNA polymerase eta (Makarova, A. V., 2012). Pol Iota has been shown to function better with manganese ions rather than magnesium ions (Makarova, A. V., 2012). The general architecture is similar to that of other proteins in this family. Like DNA polymerase eta, there is the palm region with the active site, along with a little finger and thumb domain to bind with the DNA and a fingers domain to facilitate the phosphodiester bond. DNA polymerase eta has a weaker interaction between the fingers and the little finger domain. This reduced mobility causes for increased contact between the little finger and the DNA molecule, resulting in the reaction happening with less error. BLAST comparisons of the sequences of the two proteins yielded a 42% positives match, meaning that 42% of the primary structure aligned by total identity or by charge. There is an active site for the binding of the nucleotide from Asp-30 through Phe-35 on pol eta, this was found to match with Asp-34 through Phe-38 on pol iota. Both these regions exhibit an initial beta sheet, a beta turn, and then an alpha helix. This provides a large amount of surface area to interact with the nucleotide. There was also a large similarity between Tyr-64 through Arg-67 on pol eta and Tyr-68 through Arg-71 on pol iota. These residues are also involved in binding to the free nucleotide. There are major similarities between residues Lys-211 through Val-286 on pol eta and residues Glu-147 through Leu-221. This is the palm region. This region has a large chain lacking an assigned secondary structure, followed by a large alpha helix. This allows for greater mobility, helping the protein translocate as it functions.