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Thermophilic DNA Polymerase Alpha-Subunit

Introduction

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 and elongation of the growing DNA 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. 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). 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.

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?

Components of Taq DNA Polymerase III α-Subunit

VIDEO 1 VIDEO 2

Important Domains

The fingers, palm and thumb are highly conserved domains. They are nicknamed as such because the structures resemble a closed hand. They form the nucleotide binding pocket and are involved in nucleotide positioning and sugar selection, nucleotide addition, and DNA interactions.

• Palm (residues 286-492 and 575-622) - The palm domain contains the catalytic site for nucleotide addition, including the three conserved catalytic aspartates found in all nucleotidyl transferases (CHECK INFO). The palm domain also aids in positioning incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis (base near template strand base and phosphates adjacent to catalytic aspartates).
• Fingers (residues 623-835) - The fingers domain selects for deoxyribonucleotides over ribonucleotides by evaluating the sugar group. It also aids in positioning the incoming deoxyribonucleotides so that the conjugate base is in the correct orientation for catalysis. The fingers domain also forms part of the active site.
• Thumb (residues 493-574) - The thumb domain forms part of the DNA exit channel and interacts with DNA.
• β-binding domain (residues 836-1012) - The β-binding domain binds the β-sliding clamp and also positions double-stranded DNA.
• C-terminal domain (CTD) (residues 1013-1220) - The CTD contains an oligonucleotide binding (OB) fold that binds single-stranded DNA by burying it in a surface groove.
• Polymerase and histodinol phosphatase (PHP) domain (residues 1-285) - The PHP domain forms part of the DNA exit channel. It may have proofreading function via zinc-ion dependent exonuclease activity, although this is yet to be determined.
• Incoming deoxyribonucleotide (dNTP) (residues 1221-1222) - This is the incoming deoxyribonucleotide that must undergo sugar selection, orientation and positioning, stabilization, catalysis, and addition to the nascent DNA chain.

DNA Interactions & Conformational Changes

The HhH DNA binding motif directly contact the sugar-phosphate backbone of DNA at the minor groove. Contact is made using positively-charged residues on the negatively-charged backbone. Before contact is made, the thumb and PHP domains block the positioning of double-stranded DNA. Upon contact, the PHP domain rotates away from the DNA, allowing the thumb to contact the minor groove of the DNA. The β-binding domain, fingers and thumb also adjust.

Nucleotide Addition

2012 UW-Milwaukee CREST Team

Team Members

Joseph Johnston, Bryan Landrie and Anne Marie Wannamaker

Abstract

ABSTRACT

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Bacterial RNA Polymerase: New Insights on a Fundamental Molecular Machine

Abstract

RNA Polymerase Elongation Complex

The RNAP holoenzyme is a molecular machine comprised of six subunits that copies DNA to RNA. RNAP initially binds to DNA at the promoter to form the closed complex ^[2]. The DNA surrounding the promoter sequence unwinds to form the open complex consisting of a 17 base pair transcription bubble (http://www.pingrysmartteam.com/RPo/RPo.htm) (Note: Different nomenclature is used)^[3]. The transcribed template strand is held inside the active site channel while the non-template strand is held between the rudder and clamp helices, away from the active site. RNAP releases from the promoter and transitions to the elongation complex that moves along the template strand, adding nucleotides to the 3’ hydroxyl of the RNA. The β’ subunit contains structures and forms channels that are crucial to this process.

Ribonucleotides enter through the secondary channel (15 x 20 Å)^[4]. The ribonucleotide is initially positioned at the pre-insertion site with its base forming hydrogen bonds with the template base and the triphosphate facing the active site. Subsequent movement of the ribonucleotide to the insertion site positions the triphosphate close enough to the active site for catalysis to occur^[4].

The active and secondary channels are separated by the bridge helix. Besides forming channels, the bridge helix interacts with a structure called the trigger loop, which is unstructured in this model. When a nucleotide is present, the bridge helix induces a conformation change in the trigger loop so it becomes the trigger helix^[4]. The trigger helix acts as a swinging gate while guiding ribonucleotides into their correct orientation to meet the 3’ hydroxyl of the growing RNA transcript ^[4]. The trigger helix also reduces the size of the secondary channel to 11 x 11 Å, which prevents diffusion of the complementary nucleotide away from the active site while simultaneously preventing interference from other nucleotides ^[4].

References

1. ↑ ^{1.0 1.1 1.2 1.3} Winning, R.S. (2001). "DNA Replication." Eastern Michigan University. Retrieved from http://www.emunix.emich.edu/~rwinning/genetics/replic4.htm.
2. ↑ Snyder, L. & Champness, W. (2007). Molecular genetics of bacteria (3rd ed.). Washington, D.C.: ASM Press.
3. ↑ 2006 Pingry SMART Team: RNA Polymerase Holoenzyme Open Promoter Complex (Rpo) Jmol Tutorial
4. ↑ ^{4.0 4.1 4.2 4.3 4.4} Vassylyev DG, Vassylyeva MN, Zhang J, Palangat M, Artsimovitch I, Landick R. Structural basis for substrate loading in bacterial RNA polymerase. Nature. 2007 Jul 12;448(7150):163-8. Epub 2007 Jun 20. PMID:17581591 doi:10.1038/nature05931