OCR Specification focus:
'Describe helicase and DNA polymerase roles; replication accuracy and random, spontaneous mutations.'
DNA replication ensures the faithful transmission of genetic information from one generation of cells to the next. The process is semi-conservative, producing two identical DNA molecules, each with one parental and one newly synthesized strand.
The Principle of Semi-Conservative Replication
The semi-conservative model of DNA replication was demonstrated by Meselson and Stahl (1958). Their experiment confirmed that each daughter DNA molecule consists of one original (template) strand and one newly synthesized strand.
Schematic of the Meselson–Stahl experiment showing the shift from heavy (^15N) to intermediate/light DNA bands across generations after replication in ^14N medium. The pattern of bands provides direct evidence that each new DNA molecule contains one parental and one newly synthesized strand. Extra contextual labels (e.g., generation numbers) are included but remain strictly supportive of the syllabus focus. Source.
Key Stages of Semi-Conservative Replication
DNA replication occurs during the S phase of the cell cycle and involves a precise sequence of enzymatic steps to ensure high fidelity.
Unwinding of the DNA Double Helix
The enzyme DNA helicase breaks the hydrogen bonds between complementary base pairs (A–T and C–G).
This creates two template strands, exposing the nitrogenous bases for replication.
Formation of the Replication Fork
As helicase unwinds DNA, a replication fork forms — a Y-shaped region where new strands are synthesized.
Single-strand binding proteins (SSBPs) may stabilize the separated strands, preventing them from rejoining.
Primer Binding
A short strand of RNA primer, synthesized by primase, provides a free 3’ hydroxyl group (–OH) for nucleotide addition.
This is essential because DNA polymerase can only add nucleotides to an existing strand, not initiate synthesis.
Elongation by DNA Polymerase
DNA polymerase catalyzes the condensation reaction between the 5’ phosphate of an incoming nucleotide and the 3’ hydroxyl group of the previous nucleotide, forming a phosphodiester bond.
New nucleotides are added in the 5’ → 3’ direction.
Phosphodiester bond: A covalent bond between the phosphate group of one nucleotide and the hydroxyl group of another, forming the sugar-phosphate backbone of DNA.
Leading and Lagging Strand Synthesis
The leading strand is synthesized continuously toward the replication fork.
The lagging strand is synthesized discontinuously away from the fork in short fragments known as Okazaki fragments.
These fragments are later joined by DNA ligase, sealing gaps in the sugar-phosphate backbone.
A labeled replication-fork diagram showing helicase opening DNA, RNA primers, continuous synthesis on the leading strand, and discontinuous synthesis on the lagging strand as Okazaki fragments, which are later joined by DNA ligase. The layout reinforces 5′→3′ directionality and explains why synthesis is semi-discontinuous. Some panels include a broader eukaryotic context, but all labels directly support the OCR-required mechanism. Source.
Termination and Proofreading
Once the entire molecule is replicated, DNA polymerase proofreads the new strands by checking complementary base pairing.
Incorrectly inserted nucleotides are excised and replaced, ensuring replication accuracy.
Proofreading: The error-checking process by DNA polymerase that identifies and corrects mismatched bases during DNA replication.
Enzymes Involved in Semi-Conservative Replication
DNA Helicase
Function: Unwinds the DNA helix by breaking hydrogen bonds.
Importance: Creates the replication fork, enabling complementary base pairing.
DNA Polymerase
Function: Catalyzes the formation of phosphodiester bonds between adjacent nucleotides.
Directionality: Operates only in the 5’ → 3’ direction.
Accuracy: Has intrinsic proofreading ability that significantly reduces error frequency (approximately one mistake per 10⁹ base pairs).
DNA Ligase
Function: Joins Okazaki fragments on the lagging strand.
Mechanism: Seals nicks in the sugar-phosphate backbone through condensation reactions.
Base Pairing and Replication Fidelity
Complementary base pairing is fundamental to accurate DNA replication. Adenine (A) pairs with thymine (T) through two hydrogen bonds, while cytosine (C) pairs with guanine (G) through three hydrogen bonds.
This ensures that the base sequence of the new strand is identical to that of the original.
Complementary base pairing: The specific pairing of nitrogenous bases in DNA (A with T, and C with G) via hydrogen bonds.
Errors during base pairing can lead to mutations, which are changes in the DNA sequence. Proofreading by DNA polymerase minimizes these errors, maintaining genomic integrity.
Mutations: Origin and Consequences
Nature of Mutations
Mutations are random and spontaneous changes in the DNA sequence. They may occur during replication or be induced by external agents known as mutagens (e.g., UV light, ionizing radiation, or chemical exposure).
Mutation: A change in the nucleotide sequence of DNA that may alter genetic information.
Types of Mutations
Substitution: One base is replaced by another (e.g., adenine replaced with cytosine).
Insertion: An extra base is added, shifting the reading frame (frameshift mutation).
Deletion: A base is lost, which can also potentially cause a frameshift.
Frameshift mutations are particularly harmful because they alter the triplet codon reading frame, leading to abnormal polypeptides.
Vector illustration of substitution, insertion, and deletion mutations in a short DNA segment. Insertions and deletions can produce frameshift mutations, altering downstream codons and potentially protein structure. The figure is limited to core mutation types; any additional styling is purely visual and does not add extra content. Source.
Causes of Mutations
Replication errors — occasional failure of DNA polymerase proofreading.
Spontaneous base changes — such as tautomeric shifts, where a base temporarily changes shape and pairs incorrectly.
Environmental mutagens — radiation, chemicals, or viruses that damage DNA.
Effects of Mutations
Neutral mutations: No effect on phenotype, often due to the degenerate genetic code.
Beneficial mutations: Rare changes that confer an advantage (e.g., antibiotic resistance in bacteria).
Harmful mutations: May result in non-functional proteins or disease (e.g, cystic fibrosis, sickle cell anemia).
Replication Accuracy and Repair Mechanisms
While replication is highly accurate, errors that escape proofreading can still occur. Cells possess post-replication repair mechanisms to correct mismatches:
Mismatch repair enzymes detect distortions in the DNA helix and remove mismatched bases.
The gap is filled by DNA polymerase and sealed by DNA ligase.
This maintains genetic stability across generations of cells.
Summary of Key Features of Semi-Conservative Replication
Semi-conservative: Each DNA molecule contains one parental and one new strand.
Enzyme-dependent: Helicase, polymerase, ligase, and primase coordinate replication.
Directionality: Synthesis occurs in the 5’ → 3’ direction only.
High fidelity: Proofreading ensures minimal errors.
Mutation source: Replication errors or environmental damage lead to genetic variation.
FAQ
Semi-conservative replication preserves genetic continuity because each daughter DNA molecule retains one original strand as a template.
This ensures fewer errors and allows proofreading mechanisms to detect and correct mismatches based on the existing sequence.
In contrast, conservative replication would produce a completely new double helix and retain the old one unchanged, while dispersive replication would generate DNA with mixed fragments of old and new material—both of which are less accurate and more error-prone.
DNA polymerase checks the geometry of each newly added nucleotide. When a base pair does not match the complementary base correctly, it distorts the DNA helix.
The enzyme pauses and uses its 3′ to 5′ exonuclease activity to remove the incorrect nucleotide.
A correct nucleotide is then inserted, and replication continues.
This proofreading step reduces the mutation rate dramatically, maintaining genetic stability across cell divisions.
DNA polymerase can only add nucleotides to the free 3′ hydroxyl (–OH) group of the growing strand.
If synthesis were to occur in the 3′ to 5′ direction, removing an incorrectly paired nucleotide would leave no triphosphate group for forming the next phosphodiester bond, halting replication.
Thus, the enzyme’s structure and energy requirements enforce strict 5′ to 3′ directionality, ensuring both efficiency and accuracy in DNA synthesis.
Even in the absence of mutagens, DNA can undergo spontaneous chemical changes that alter base pairing.
Common examples include:
Depurination: loss of a purine base (adenine or guanine).
Deamination: conversion of cytosine to uracil, leading to incorrect base pairing.
Tautomeric shifts: temporary rearrangements of electrons within bases, causing mispairing during replication.
These spontaneous events are rare but significant sources of genetic variation.
Immediately after replication, the newly synthesised DNA strand lacks certain chemical modifications present on the parental strand, such as methylation.
Mismatch repair enzymes use this difference to identify which strand to correct.
They remove the mismatched section from the unmethylated (new) strand and then fill the gap using DNA polymerase and DNA ligase.
This strand discrimination is crucial for maintaining replication fidelity and preventing permanent mutations.
Practice Questions
Question 1 (2 marks)
Explain the role of DNA helicase in semi-conservative DNA replication.
Mark scheme:
1 mark: DNA helicase breaks the hydrogen bonds between complementary base pairs.
1 mark: This separates the two DNA strands, allowing each to act as a template for replication.
Question 2 (5 marks)
Describe how DNA is replicated by the semi-conservative mechanism, including the roles of the enzymes involved.
Mark scheme:
1 mark: DNA helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds.
1 mark: Each strand acts as a template for the formation of a new complementary strand.
1 mark: DNA polymerase adds complementary nucleotides to the exposed bases on the template strand, forming phosphodiester bonds.
1 mark: The leading strand is synthesised continuously; the lagging strand is synthesised discontinuously as Okazaki fragments.
1 mark: DNA ligase joins the Okazaki fragments to complete the sugar-phosphate backbone.
Accept other clear references to the 5′ to 3′ direction of synthesis or proofreading by DNA polymerase as alternative valid detail for one mark.
