DNA polymerases

DNA polymerase enzyme that synthesizes DNA by assembling nucleotides. DNA polymerase was purified by Arthur Kornberg in 1955 from E. coli cells now known as DNA polymerase I In 1959; Kornberg was awarded with Noble Prize in physiology and medicine. DNA synthesis occurs by phosphoryl group transfer. 3ˈ-OH group is nucleophile and attacks α-phosphorous of the incoming deoxynucleoside 5ˈ-triphosphate. The reaction is

(dNMP)n + αNTP → (dNMP)n + 1 + PPi

  • DNA polymerases require a template.
  • Polymerases require a primer. A primer is a strand segment with a free 3ˈ-OH to which a nucleoside can be added. Primer terminus is the free 3ˈ-OH group. Most primers are RNA oligonucleotides rather than DNA.
  • DNA polymerase either dissociates or moves along the template and adds nucleotide after adding nucleotides to the growing DNA strand.

E.coli has 5 DNA polymerases

DNA Pol I is the most abundant polymerase and is encoded by the pol A gene. It catalyzes the nucleotide polymerization in a 5´→3´ direction. This repair enzyme is involved in the processing of Okazaki fragments generated during lagging strand synthesis. DMA pol I (Kornberg enzyme) possesses four enzymatic activities.

  • 5ˈ-3ˈ (forward) DNA-dependent polymerase activity requiring 3ˈ primer site and a template strand.
  • 3ˈ-5ˈ (reverse) exo-nuclease activity that mediates to proofreading.
  • 5ˈ-3ˈ (forward) exo-nuclease activity mediating nick translation during DNA repair.
  • 5ˈ-3ˈ (forward) RNA dependent DNA polymerase activity.

DNA Pol II  is a family B polymerase. DNA Pol II possess 3’ to 5’ exonuclease activity and helps in DNA repair. DNA Pol II block Pol III activity and replicate DNA if it is damaged.

DNA Pol III It is a primary enzyme known as Holoenzyme having molecular weight 900 KD. It is the main and most active enzyme involved in DNA replication in E. coli. It consists of three assemblies i.e. the Pol III core, the beta sliding clamp processivity factor and the clamp-loading complex. Core subunits α-polymerase activity hub, ζ-exonucleolytic proofreader, θ-stabilizer for

DNA Pol IV is a γ family DNA polymerase, have no proofreading ability. It is involved in DNA mutagenesis.

DNA Pol V is a γ family DNA polymerase, have no proofreading ability. It is involved SOS response and translesion synthesis DNA repair mechanism.

Eukaryotic DNA Polymerases

DNA Pol III It is main enzyme responsible for replication. In eukaryotes, it has 10 subunits i.e., λ, β, γ, δ, δ‘, ε, θ, Ʈ, ϰ, Ѱ, where γ, δ complex (γ, ϰ, δ, δ‘, Ѱ) is sliding clamp loader, β subunit is sliding clamp. The λ-subunit have 5ˈ→3′ polymerase activity, Ʈ subunit is basically dimerization unit, ε subunit have 3ˈ→5ˈexo-nuclease activity so perform proofreading. The subunits λ, ε, θ are core enzyme.

Enzymes and proteins involved in DNA replication Replisome or DNA replicase system is the complex required for replication consisting of 20 or more different enzymes of proteins.

Helicases are basically hexamer enzymes which move along the DNA and separate the strands, using chemical energy from ATP.

Topoisomerases relieves the topological stress in the helical DNA structure, which is created after strand separation. Due to helicase action, DNA strand get some topological constrains and Topoisomerase resolve these constrains.

Topoisomerase I creates single-stranded nick, need no ATP, change linking number by 1.

Topoisomerase II needs ATP, create double-stranded nick, change linking number by 2. This is also known as Gyrase. Nalidixic Acid and Ciproflaxin inhibit topoisomerase activity. Novobiocin inhibits binding of ATP to Topoisomerase II. FEN-1 (flap endonuclease) During replication, the RNA primer is degraded by the 5ˈ-3ˈ exonuclease activity of FEN-1.

DNA Binding Proteins

SSB (Single Strand Binding) proteins stabilize the single stretch of DNA.

Primases are the enzymes, which play role in synthesizing RNA primers, which must be present on the template for synthesis of new DNA strand. It provides the initial 3ˈOH on which DNA polymerase can act.

Ligases are the enzymes, which seal the nick and remains in DNA backbone after RNA primer is removed and filled the gap with in the DNA. It forms the final phosphodiester bond. E. coli ligase uses energy of NAD.