The primary function of DNA polymerase is to replicate the DNA of an organism. DNA replication occurs just prior to cell division and is necessary for growth and reproduction of a living organism (see DNA Replication, Transcription and Translation). DNA consists of two strands of hydrogen-bonded deoxyribonucleotides (dNTPs) running anti-parallel to each other in a double helix. The deoxyribonucleotides that make up DNA consist of a nitrogen base, a deoxyribose sugar group, and a phosphate. The DNA backbone is composed of sugar groups and phosphates joined with phosphodiester bonds, giving it a negative charge. The nitrogen bases of the deoxyribonucleotides extend into the helix and form Watson-Crick base pairs (A-T and C-G) stabilized by hydrogen bonds. The result is double-stranded DNA.
The replisome is an enzymatic complex with multiple subunits (see Replisome Diagram). The core DNA polymerase subunits are α, ε, and θ, which are adjacent to the the β-clamp. This tutorial will focus on the α-subunit which contains the catalytic site for dNTP addition to the primer strand.
Prokaryotic DNA Polymerases
Several differences exist between replication in prokaryotes and eukaryotes. Eukaryotic DNA is complexed with histones that must be removed and replaced during each round of replication[1]. Organelles within the eukaryotic cell, such as mitochondria, may contain DNA that also must be replicated [1]. Prokaryotic chromosomes are circular, whereas eukaryotic chromosomes are linear[1]. The increased complexity of eukaryotic DNA replication has resulted in at least five DNA polymerases being discovered thus far[1].
Prokaryotes, on the other hand, utilize three DNA polymerases. DNA polymerases I and II are primarily involved in DNA repair, specifically in using their 3'-5' exonuclease activity to remove faulty base pairs [1]. DNA polymerase III is the main replicative polymerase in bacteria. The DNA polymerase III α-subunit shown below is that of Thermus aquaticus, commonly referred to as Taq. This crystallized structure of the α-subunit of Taq DNA polymerase III contains DNA, DNA polymerase III, and an incoming dNTP (referred to as the ternary complex)[2]. Taq DNA polymerase III, a replicative polymerase, is homologous to that of Escherichia coli and also Polβ, a eukaryotic polymerase specializing in repair instead of replication [2].
Learning Objectives
How does the α subunit of DNA polymerase III contact DNA?
Which domains select for deoxyribonucleotides?
Which domains form the catalytic site?
Which domains form the DNA exit channel?
Challenge Questions
COMING SOON
Components of Taq DNA Polymerase III α-Subunit
Important Domains
The fingers, palm and thumb domains of DNA polymerase III are highly conserved [3]. They are nicknamed as such because the structures resemble a closed hand gripping double-stranded DNA. Typically the fingers interact with the incoming deoxyribonucleotide, the palm positions catalytic ions, and the thumb holds the DNA template [4].These domains form binding pockets for both DNA and incoming dNTPs[2]. They are also involved in correct dNTP selection, position and addition along with DNA interactions [2].
(residues 286-492 and 575-622, in light green) - The palm domain contains the catalytic site for nucleotide addition, including three highly conserved aspartates [2]. The palm domain also aids in positioning the 3' primer hydroxyl group and incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (i.e., the incoming dNTP base near the primer base and triphosphate adjacent to the catalytic aspartates)[2].
(residues 623-835, in light blue) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group[5]. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis[2]. The fingers domain forms part of the active site[2].
(residues 493-574, in salmon) - The thumb domain forms part of the DNA exit channel and contains many residues that interact with the DNA[2].
(residues 836-1012, in light purple) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA[2].
(residues 1013-1220, in gray) - The CTD contains an oligonucleotide binding-fold (OB-fold) that buries single-stranded DNA in a surface groove[2].
(residues 1-285, in gray) - The PHP domain forms part of the DNA exit channel[2]. It may have proofreading function via Zn2+-dependent exonuclease activity, although this is yet to be determined[6].
- This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain[2].
(residues 463, 465 and 618) - These aspartate residues are highly conserved and essential to catalysis [2][7].
DNA Interactions & Conformational Changes
The thumb domain uses multiple positively-charged residues within two α-helices to interact with the DNA substrate at the minor groove (Taq500-511 and Taq515-526)[2]. The β-binding domain interacts with DNA via its helix-hairpin-helix (HhH) motif (Taq892-910) and adjacent loops (Taq846-852 and Taq923-927)[2]. The HhH motif directly contacts the sugar-phosphate backbone of DNA at the minor groove[2]. Contact is made using positively-charged residues on the negatively-charged backbone[2]. The PHP domain may contact the DNA at a loop [2].
Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA[2]. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA[2]. The β-binding domain, fingers and thumb also adjust[2].
evaluate sugar groups on incoming nucleotides and select for dNTPs[5].
If a ribonucleotide or an incorrectly matched dNTP enters the nucleotide binding pocket, misalignment occurs and catalysis cannot happen.
Highly conserved arginine residues interact with the triphosphate of the incoming dNTP [5][2]. These arginine residues facilitate base pairing between the primer base and dNTP through orientation of the nucleotide[5][2].
The incoming dNTP is also coordinated by (Taq G425-S426) via interactions with the γ-phosphate of the dNTP[2]. The GS motif is absolutely conserved[8].
The 3' end of the primer strand is positioned by formation of a salt bridge between [2].
Catalysis
All polymerases employ a two metal ion mechanism for catalysis[9].
One metal ion is found near the conserved aspartate residues and incoming dNTP triphosphate, while the is coordinated by the 3' primer hydroxyl group and the α-phosphate of the incoming dNTP[2].
The 3' hydroxyl group attacks the α-phosphate of the incoming dNTP. A phosphodiester bond is formed between the 3' hydroxyl of the primer strand and the 5' α-phosphate group of the incoming dNTP. A pyrophosphate consisting of the incoming dNTP's β- and γ-phosphates is released.
2012 UW-Milwaukee CREST Team
Model Researchers and Designers
Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker
↑ Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem. 1999 Jun 18;274(25):17395-8. PMID:10364165
↑ 5.05.15.25.3 Bailey S, Wing RA, Steitz TA. The structure of T. aquaticus DNA polymerase III is distinct from eukaryotic replicative DNA polymerases. Cell. 2006 Sep 8;126(5):893-904. PMID:16959569 doi:10.1016/j.cell.2006.07.027
↑ Stano NM, Chen J, McHenry CS. A coproofreading Zn(2+)-dependent exonuclease within a bacterial replicase. Nat Struct Mol Biol. 2006 May;13(5):458-9. Epub 2006 Apr 9. PMID:16604084 doi:10.1038/nsmb1078
↑ Pritchard AE, McHenry CS. Identification of the acidic residues in the active site of DNA polymerase III. J Mol Biol. 1999 Jan 22;285(3):1067-80. PMID:9887268 doi:10.1006/jmbi.1998.2352
↑ Aravind L, Koonin EV. DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history. Nucleic Acids Res. 1999 Apr 1;27(7):1609-18. PMID:10075991
↑ Steitz TA, Smerdon SJ, Jager J, Joyce CM. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. Science. 1994 Dec 23;266(5193):2022-5. PMID:7528445
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