Pol α DNA Polymerase
Created by Lana Le
Introduction
Deoxyribonucleic acid (DNA) polymerases are classified into seven families based on sequence homology: A, B, C, D, X, Y and RT. Pol α DNA polymerase, found in a variety of organisms from prokaryotes to eukaryotes, is part of Family B and is involved in DNA repair and replication. Pol α DNA polymerase plays an important role in the temporary complex where the leading strand and lagging strand synthesis occurs (1). Pol α DNA polymerase from Enterobacteria phage RB69 (PDB ID: 1WAF), also known as gp43, catalyzes the addition of a deoxynucleoside triphosphate to a chain of DNA in synthesis of DNA as well as possesses an exonucleolytic activity that degrades single stranded DNA in the 3’-to-5’ direction. The protein has a molecular weight of 209,208.55 Da and an isoelectric point of 5.77.
Overall Structure
The structure of pol α DNA polymerase from Enterobacteria phage RB69 is related to its function. Regarding to its secondary structure, the protein has 33 alpha helices and 26 beta sheets. Overall, its shape could be thought of as a disk with a hole in the center. The hole is the polymerase active site, and rising from this hole are three deep grooves constituted by five domains: the palm, the thumb, the finger, the exonuclease, and the NH2-terminal domains. The palm domain contains the polymerase active site, the thumb domain interacts with the duplex DNA product, the fingers domain interacts with the template strand, and the exonuclease domain binds the primer in the editing mode (1).
Hydrogen bonds
Gp43 has two chains, A and B, which interact with each other through non-bonded contacts and hydrogen bonds. The residues involved in hydrogen bonding are Tyr-776 and lle-78, Leu-857 and Lys-55, Lys-859 and Gly-79, Pro-155 and Val-436, Gln-318 and Gln-389, Asn-316 and Gln-389, and Asp-860 and Glu-81.
Functionally Important Residues
As a member of the Family B DNA polymerase, gp43 also contains the family signature sequence: YGDTDSIYV. There are also many other important residues in gp43 that are conserved among pol α DNA polymerases. Highly conserved regions converge at the center of the active site to generate a contiguous conserved surface. These conserved regions can be characterized into three chemically distinct groups: aromatic residues Tyr-416, Tyr-567, and Tyr-391; negatively charged residues Asp-621, Asp-623, Asp-411, Asp-684, and Glu-686; and positively charge residues Lys-560, Arg-482, and Lys-486. Among these residues, the highly conserved aspartic acid residues Asp-411, Asp-621 and Asp-623 (view space model), clustered on three β strands of the polymerase palm domain, hold functional significance because their mutations result in loss of activity of the polymerase (2).
Similar proteins
The N-terminus of RB69 gp43 can be separated from the C-terminus and itself contains two domains (NH2 and Exo). The N-terminus of pol α DNA polymerase from Enterobacteria phage RB69 has a very similar primary structure to its homolog (PDB ID: 1NOZ) from T4 phage (3) as well as other pol α family polymerases. The sequence similarity between RB69 gp43 and T4 gp43 is 63% (BLAST E-value = 6 x 10-163), and their Z-score in the Dali test (test of 3D structural similarity) is 33.5. The exonuclease editing domains in RB69 and T4 both possess the 3’-5’ exonuclease activity. However, in their secondary structure composition, they are different in the numbers of beta strands, alpha helices, helix-helix interactions, beta turns, and gamma turns. RB69 gp43 folds most differently than its homolog from T4 at beta strand 4 (residues 33-42), strand 5 (residues 54-63), and alpha helix A (residues 64-78). Moreover, T4 DNA Polymerase fragment has one disulfide bond while gp43 does not.
Several other DNA polymerases also share structural and functional similarities with pol α family polymerase from Enterobacteria phage RB69. DNA polymerases from E. coli Klenow fragment (PDB ID: 1KLN) (4), Thermus aquaticus (PDB ID: 1TAU) (5), and HIV transcriptase (PDB ID: 2HMI) (6) also show a common shape with the palm, finger and thumb domains. Similar to what is observed in pol α family polymerase in Enterobacteria phage RB69, the thumb domain binds with the product duplex DNA, the palm contains the polymerase catalytic site, and the finger binds with the template strand in these comparison proteins.
Binding Pocket
During DNA replication, oxidized guanosine often rotates the sugar phosphate backbone to expose the N-7 for bonding (7).
Biological Significance
The study of pol α DNA polymerase is significant in several biological fields. Before cells can divide and grow, they have to replicate their DNAs; cells enter senescence when their DNAs stop replicating. Cell growth depends on the expression of a set of genes that specify cellular proteins, some of which participate in DNA replication. Thus deregulation of this expression plays a crucial part in the ageing process. Pol α DNA has the ability to initiate de novo DNA synthesis and is also required for the initiation for the synthesis of the leading strand as well as the lagging strand (8). Pol α DNA polymerase also interacts with other regulating proteins to control when the cell can enter the S phase in the cell cycle (8). The regulation of pol α DNA polymerase may ultimately lead to the failure of old cells to make the transition from G1 to S phase (8). As a result of its role in DNA replication and cell growth, pol α DNA polymerase continues to be a frequently studied area in cellular ageing and cancer research.