DNA Replication

Part 2 - DNA Replication

DNA Definition

• DNA stands for deoxyribonucleic acid.

Replication Process

Replication Fork

• Description: Two replication forks moving in opposite directions on a circular chromosome.

• Size: Approximately 1 μm.

Characteristics of Replication Forks

• Speed: Individual replication forks operate at approximately 1000 base pairs (bp) per second.

• Completeness: Every base pair in the genome is replicated during this process.

• Accuracy: The error rate is about one in 10^10 bp, meaning that errors are extremely rare.

• Okazaki Fragments:

  • These are short DNA segments formed on the lagging strand during replication.

  • Size in Bacteria: Approximately 1,000 to 2,000 nucleotides long.

  • Size in Eukaryotic Cells: Approximately 150 to 200 nucleotides long.

  • Synthesis of Okazaki fragments is followed by splicing the fragments together using DNA ligase.

DNA Strands Orientation

• Template Strand: Runs in a 5' to 3' direction, denoted with hydroxyl (−OH) at the 3' end.

• Primer Strand: Also runs in a 5' to 3' direction.

• Nucleotide Incorporation: Correct positioning is essential. A deoxyribonucleoside triphosphate (dNTP) must be base-paired correctly to facilitate the reaction's progression.

• Growth Direction: DNA polymerase synthesizes new strands in the 5'-to-3' direction.

  • Correct base-pair geometry must be maintained for successful elongation.

Replication Fork in Bacteria

• The DSM refers to origin of replication (ORI). This site initiates bidirectional replication of circular DNA.

• Replication Speed Comparison:

  • Bacteria: 1,000 bp/s

  • Eukaryotes: 50 bp/s

Semiconservative DNA Replication

• Semiconservative Model: This highlights that each new DNA molecule comprises one parental (template) strand and one newly synthesized strand.

Meselson-Stahl Experiment

• Experiment Overview:

  • Cells were initially grown in a medium with heavy nitrogen (15N) and later transferred to a medium with light nitrogen (14N).

  • After one generation, DNA was extracted and settled at a higher density in the gradient, indicating the incorporation of both nitrogen isotopes.

  • After a second generation, two hybrid DNAs and two light DNAs were produced, shaping understanding of semiconservative replication.

DNA Polymerase Functionality

Binding and Mechanism

• DNA polymerase binds at the primer-template junction and facilitates the synthesis of new DNA strands.

• The polymerase interacts primarily with the minor groove of DNA.

  • Major Groove Interactions: Noted patterns of hydrogen donors and acceptors (e.g., HDAA, ADAM).

  • Minor Groove Interactions: Non-sequence specific interactions (e.g., ADA, AHA).

Construction of DNA

• Nucleotide Triphosphates (NTPs): Each nucleotide added during DNA synthesis must position its alpha-phosphate and 3’-OH correctly.

• Irreversibility of Reaction: The cleavage of pyrophosphate during nucleotide addition augments driving the reaction forward, allowing the DNA strand to extend by one nucleotide.

Proofreading Mechanism

• DNA Polymerase Accuracy: Typically makes 1 mistake per 10^5 bp synthesized without proofreading.

  • Proofreading enhances accuracy to one mistake per 10^7 bp.

• Tautomer Misalignment: Temporary forms, such as the enol form of guanine mimicking adenine, can lead to incorrect base pairing during replication.

Summary of DNA Polymerase Functions

1. Requires a 3' OH primer for initiation.

2. Binds to longer single-stranded DNA (ssDNA) for template utilization.

3. The added dNTP must correctly base-pair for catalysis.

4. Extends the 3' OH of the primer through DNA synthesis.

5. Works with a single active site for nucleotide addition.

6. Does not differentiate between different nucleotide types as base pairs fit universally within geometric parameters.

7. Correct base pairing positions the 3’ OH near the α-phosphate of the dNTP, crucial for catalysis.

Comparison of DNA Polymerases in E. coli

Table: Comparison of DNA Polymerases

Subunits of DNA Polymerase III

Table: Subunits Overview

Replication Fork Machinery

• During replication, an enzyme called DNA primase synthesizes RNA primers at intervals for Okazaki fragments.

Continuous Synthesis on Leading Strand

• Leading Strand Synthesis: Occurs continuously as DNA is unwound by the helicase DnaB.

  • The clamp-loading complex acts on the leading strand enabling DNA polymerase to function smoothly.

Lagging Strand Dynamics

• The lagging strand undergoes discontinuous synthesis due to the necessity for Okazaki fragments that are later ligated by DNA ligase.

DNA Helicase and Single-Stranded Binding Proteins

• DNA Helicase: Binds to separate strands of DNA by hydrolyzing ATP, functioning like a rotary engine to facilitate the unwinding process.

• Single-Stranded Binding Proteins (SSB): Stabilize single-stranded regions of DNA during replication, preventing hairpin formation and re-annealing.

Topoisomerase Functions

• Topoisomerase I: Relieves supercoiling tension ahead of the replication fork by creating transient single-strand breaks that allow the DNA to rotate freely.

DNA Repair Mechanisms

Enzymes/Proteins Involved

Methyl-directed Mismatch Repair

• Distinction between parent and newly synthesized strands involves methylation at specific sequences (5'-GATC-3') in E. coli. MutH cleaves at the unmethylated strand to initiate repair.

Types of Double-Strand Breaks Repair

Mechanisms of Repair

• Nonhomologous End Joining: Quick repair that can result in deletions at the break site.

• Homologous Recombination: Accurate repair using the sister chromatid as a template for repair.

Telomere Replication and Repair

• Telomerase: Extends the DNA at the telomeric ends to maintain chromosome integrity during replication.

  • The enzyme utilizes an RNA template for synthesis of the repetitive DNA sequences.

DNA Damage Mechanisms

• Depurination and Deamination: Common spontaneous reactions that lead to various DNA damages.

  • Depurination refers to the loss of purine bases (adenine, guanine) from the DNA.

  • Deamination converts cytosine into uracil, disrupting normal base-pairing.

Inherited Human Syndromes with DNA Repair Defects

Syndromes and Effects

• Xeroderma pigmentosum (XP): Involves defects in nucleotide excision repair, leading to skin cancer and UV sensitivity.

• Ataxia telangiectasia: Associated with DNA double-strand break repair defects; leads to neurological abnormalities and cancer risks.

• BRCA1 and BRCA2: Implicated in breast and ovarian cancer risks due to homologous recombination repair deficiencies.