User:Catherine L Dornfeld/Sandbox 1

From Proteopedia

Jump to: navigation, search


Alpha Subunit of Thermus aquaticus DNA Polymerase III


Role of DNA Polymerase in Replication

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


Components of Taq DNA Polymerase III α-Subunit

Videos Detailing Functional Core and Active Site

<swf width="500" height="330"></swf> <swf width="500" height="330"></swf>

DNA Polymerase III Alpha Subunit (PDB entry 3e0d)

Drag the structure with the mouse to rotate

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].

Nucleotide Addition

DNA polymerase III α-subunit (PDB entry 3e0d)

Drag the structure with the mouse to rotate

2012 UW-Milwaukee CREST Team

Model Researchers and Designers

Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker




  1. 1.0 1.1 1.2 1.3 1.4 Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from
  2. 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21 2.22 2.23 2.24 2.25 2.26 2.27 Wing RA, Bailey S, Steitz TA. Insights into the replisome from the structure of a ternary complex of the DNA polymerase III alpha-subunit. J Mol Biol. 2008 Oct 17;382(4):859-69. Epub 2008 Jul 27. PMID:18691598 doi:10.1016/j.jmb.2008.07.058
  3. Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783-90. PMID:1377403 doi:[
  4. Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem. 1999 Jun 18;274(25):17395-8. PMID:10364165
  5. 5.0 5.1 5.2 5.3 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
  6. 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
  7. 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
  8. 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
  9. 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

Proteopedia Page Contributors and Editors (what is this?)

Catherine L Dornfeld

Personal tools