07-Biochemistry-Lecture7 | Week 4 - Lecture 2

What is DNA Metabolism?

• Stable storage of genetic information, yet dynamic.

• Key processes:

  • DNA Replication: High fidelity synthesis before cell division.

  • DNA Repair: Error detection and correction post-synthesis.

  • DNA Recombination: Rearranging DNA segments between molecules for genetic diversity.

• DNA metabolism includes tightly regulated processes and utilizes DNA as a substrate.

Key Topics in DNA Metabolism

• DNA replication in bacteria

• DNA replication in eukaryotes

DNA Replication in Bacteria

• DNA elongation chemistry:

  • Parental DNA Strand: Acts as a template.

  • Nucleoside Triphosphates (dNTPs): Substrates for strand synthesis.

  • Mechanism: 3’-OH group attacks the a-phosphate of incoming trinucleotide with Mg2+ ions assisting.

  • Elongation Equation: (dNMP)n + dNTP → (dNMP)n+1 + PPi.

Mechanism of DNA Polymerases

• Catalytic Mechanism: Involves 2 Mg2+ ions for nucleotide addition; facilitates transition states.

• Processivity: Refers to the number of dNTPs added before dissociation.

• Requirements: Template, primer, and dNTPs.

Primer Features

• Primer Characteristics:

  • Short complementary strand with a free 3’-OH group.

  • Must be either DNA or RNA.

  • Each incoming nucleotide selected by base-pairing to the template.

Structural Features of DNA Polymerases

• DNA Polymerase Structure: Pocket with two regions (insertion and post-insertion sites).

• Shape resembles a human hand wrapping around the active site.

DNA Polymerase Activity

• Adds nucleotides or dissociates; processivity varies widely among polymerases.

High Fidelity of DNA Synthesis

• Geometry of Base Pairing: Active site excludes incorrect geometries; proofreading capabilities present.

• Error rates:

  • 1 in 10^5 during polymerization.

  • Combined error rate ~1 in 10^10 due to multiple correction steps.

Proofreading Mechanisms

• 3’→5’ Exonuclease Activity: Proofreads and corrects mismatches by removing incorrect nucleotides.

• Mechanism of proofreading improves accuracy significantly.

Various DNA Polymerases in E. coli

• Polymerase I: Slow and low processivity; mainly involved in cleanup.

• Polymerase III: Main replication polymerase with high rate and processivity.

• Polymerases II, IV, V: Mainly involved in DNA repair.

5’→3’ Exonuclease Activity of DNA Polymerase I

• Nick translation removes nucleotides ahead of the enzyme, allowing movement along the DNA strand.

Architecture of DNA Polymerase III

• Made up of 10 subunits with a clamp-loader complex for processivity >500,000 bp.

E. coli Replisome Components

• Includes helicases, primases, DNA polymerases, DNA ligases, and gyrases for replication.

Initiation of Replication in E. coli

• Begins at Origin oriC: Contains critical sequence elements that facilitate binding and unwinding.

Role of DnaA Proteins

• Bind at R and I sites, causing DNA to wrap around and supercoil, leading to strand separation.

DnaB Helicase Function

• Unwinds DNA strands in preparation for synthesis, employing SSB to stabilize separated strands.

Regulation of Initiation

• Hda promotes dissociation of the DnaA complex, ensuring replication initiation occurs once per cycle.

Elongation Phase of Replication

• Leading and lagging strand synthesis occurs simultaneously through coordinated action within the replisome.

Transitioning Between Okazaki Fragments

• Polymerase III may switch between different Okazaki fragments as needed, with DNA ligase sealing nicks afterward.

Final Steps of Lagging Strand Synthesis

• RNA primers are replaced with DNA, and nicks are sealed by DNA ligase.

Termination of Replication

• Replication forks terminate at specific Ter sequences, and replication is separated by topoisomerases.

DNA Replication in Eukaryotes

• More complex than in bacteria with multiple origins of replication and unique regulatory mechanisms.

Eukaryotic Initiation Requirements

• Involves the Origin Recognition Complex (ORC) and is tightly regulated by cyclins and CDKs.

Multiple DNA Polymerases in Eukaryotes

• Diverse roles similar to those in bacteria, including specialized polymerases for DNA repair and replication.

Other Eukaryotic Proteins in Replication

• Include RPA (SSB equivalent) and RFC (clamp loader).

Eukaryotic Replication Speed

• Slower synthesis rate compensated by multiple origins, overall providing efficiency in genomic replication.