D1.1 DNA Replication

D1.1 DNA Replication

Overview

DNA replication is the process by which a cell makes an identical copy of its DNA before cell division. This ensures that genetic information is accurately passed from parent cells to daughter cells, and from one generation to the next.

Replication is semi-conservative — each new DNA molecule contains one original (parental) strand and one newly synthesised strand.

Why DNA Replication is Necessary

DNA Structure Review

Understanding replication requires knowledge of DNA structure:

Nucleotide Components

Each nucleotide consists of:

1. Deoxyribose sugar (5-carbon sugar)

2. Phosphate group (PO₄³⁻)

3. Nitrogenous base (one of four types)

The Four Bases

Complementary base pairing rules:

• A always pairs with T (2 hydrogen bonds)

• G always pairs with C (3 hydrogen bonds)

• Purine always pairs with pyrimidine (maintains constant width)

Double Helix Structure

Strand Directionality

DNA strands have directionality based on carbon numbering in deoxyribose:

• 5' end — Phosphate group attached to 5' carbon

• 3' end — Hydroxyl group (-OH) attached to 3' carbon

• DNA polymerase can only add nucleotides to the 3' end

• Synthesis always proceeds 5' → 3'

5' end                                           3' end
    ↓                                               ↓
Phosphate—Sugar—Base · · · Base—Sugar—Phosphate
                    ↑ ↑ ↑
              Hydrogen bonds
                    ↓ ↓ ↓
Phosphate—Sugar—Base · · · Base—Sugar—Phosphate
    ↑                                               ↑
3' end                                           5' end

Semi-Conservative Replication

The Hypothesis

Watson and Crick proposed that DNA replication is semi-conservative:

• The two strands separate

• Each strand serves as a template for a new strand

• Each daughter DNA molecule has one old strand and one new strand

Alternative Hypotheses

The Meselson-Stahl Experiment (1958)

The most elegant experiment in molecular biology — provided definitive proof of semi-conservative replication.

Materials and Methods

1. Grow E. coli in heavy nitrogen (¹⁵N) medium

   • Bacteria incorporate ¹⁵N into DNA bases

   • DNA becomes "heavy" (higher density)

2. Transfer to light nitrogen (¹⁴N) medium

   • New DNA synthesised uses ¹⁴N

   • New DNA is "light" (lower density)

3. Extract DNA at intervals (after 0, 1, 2+ generations)

4. Separate by density using caesium chloride (CsCl) gradient centrifugation

   • DNA settles at position matching its density

   • Heavy DNA sinks lower; light DNA floats higher

Results

Conclusions

• Conservative model ruled out — No heavy band after generation 1

• Dispersive model ruled out — Two distinct bands after generation 2 (not one intermediate)

• Semi-conservative model confirmed — Results exactly match predictions

Generation 0:    ═══════════ Heavy (¹⁵N-¹⁵N)

Generation 1:    ─────────── Intermediate (¹⁵N-¹⁴N)
                 ─────────── Intermediate (¹⁵N-¹⁴N)

Generation 2:    ─────────── Intermediate (¹⁵N-¹⁴N)
                 - - - - - - Light (¹⁴N-¹⁴N)
                 ─────────── Intermediate (¹⁵N-¹⁴N)
                 - - - - - - Light (¹⁴N-¹⁴N)

The Replication Process

DNA replication involves many enzymes and proteins working together at the replication fork.

Overview of Steps

1. Initiation — Replication begins at origin of replication

2. Unwinding — Double helix is unwound and strands separated

3. Primer synthesis — RNA primers provide starting point

4. Elongation — New strands synthesised by DNA polymerase

5. Primer removal and replacement — RNA primers replaced with DNA

6. Ligation — Gaps sealed to create continuous strands

7. Termination — Replication completes when forks meet

Key Enzymes and Their Functions

Detailed Step-by-Step Process

Step 1: Initiation

• Replication begins at specific sequences called origins of replication (ori)

• Prokaryotes: single origin

• Eukaryotes: multiple origins (allows faster replication of larger genomes)

• Initiator proteins recognise and bind to origin

• DNA is locally unwound to form replication bubble

Step 2: Unwinding and Stabilisation

Helicase:

• Binds to origin

• Uses ATP energy to break hydrogen bonds

• Separates (unzips) the two DNA strands

• Creates replication fork — Y-shaped region where replication occurs

Single-strand binding proteins (SSBPs):

• Bind to exposed single strands

• Keep strands separated

• Prevent nuclease degradation

• Prevent secondary structure formation

Topoisomerase/Gyrase:

• Works ahead of the replication fork

• Relieves positive supercoiling (overwinding tension)

• Cuts one or both strands, allows rotation, then rejoins

• Essential — without it, DNA would become too tightly wound to replicate

Step 3: Primer Synthesis

Why primers are needed:

• DNA polymerase cannot initiate synthesis de novo

• Can only ADD nucleotides to an existing 3'-OH group

• Requires a free 3' end to begin synthesis

Primase:

• Synthesises short RNA primers (8–12 nucleotides)

• Complementary to template strand

• Provides 3'-OH for DNA polymerase to extend

• Multiple primers needed on lagging strand

Step 4: Elongation

DNA Polymerase III (main replicative enzyme):

Key properties:

1. Requires template — Reads template strand 3'→5'

2. Requires primer — Needs free 3'-OH to start

3. 5'→3' synthesis — Only adds nucleotides to 3' end

4. Complementary base pairing — Adds correct nucleotide opposite template base

5. Proofreading (3'→5' exonuclease activity) — Checks each nucleotide; removes mismatches

Mechanism:

1. Incoming deoxyribonucleoside triphosphate (dNTP) enters active site

2. Base pairs with template base

3. Phosphodiester bond forms between 3'-OH and 5'-phosphate

4. Two phosphates released (provides energy)

5. Polymerase moves to next position

6. Process repeats (~1000 nucleotides/second in E. coli)

The Problem of Antiparallel Strands

Because the two template strands run in opposite directions, but DNA polymerase only works 5'→3', the two new strands are synthesised differently:

Leading Strand Synthesis

• Template runs 3'→5'

• New strand synthesised 5'→3' toward the replication fork

• Continuous synthesis — single primer, uninterrupted

• Only one RNA primer needed

• DNA polymerase follows helicase smoothly

Lagging Strand Synthesis

• Template runs 5'→3'

• New strand synthesised 5'→3' away from the replication fork

• Discontinuous synthesis — multiple primers, synthesised in fragments

• Called Okazaki fragments (1000–2000 nucleotides in prokaryotes; 100–200 in eukaryotes)

• Each fragment requires its own RNA primer

• Fragments must be joined together later

                    Direction of fork movement →
                            
    3'─────────────────────────────────────────5' (Template)
       ←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←←
    5'═════════════════════════════════════════3' (Leading strand)
                    ↑ Continuous synthesis
    
    5'─────────────────────────────────────────3' (Template)
       →→→   →→→→   →→→→→   →→→→→   →→→→→→
    3'═══   ════   ═════   ═════   ══════════5' (Lagging strand)
       ↑      ↑       ↑       ↑       ↑
    Okazaki fragments (discontinuous synthesis)

Step 5: Primer Removal and Replacement

DNA Polymerase I:

• Removes RNA primers using 5'→3' exonuclease activity

• Simultaneously fills gaps with DNA nucleotides

• Works in 5'→3' direction

• Cannot seal final gap between fragments

Step 6: Ligation

DNA Ligase:

• Joins Okazaki fragments on lagging strand

• Forms phosphodiester bond between 3'-OH of one fragment and 5'-phosphate of the next

• Uses ATP (eukaryotes) or NAD⁺ (prokaryotes) as energy source

• Creates continuous sugar-phosphate backbone

Step 7: Termination

In prokaryotes (E. coli):

• Two replication forks meet at terminus region

• Tus proteins and ter sequences halt replication

• Topoisomerase separates the two daughter molecules

In eukaryotes:

• Multiple forks from multiple origins meet

• Replication ends when all forks complete

• Special consideration for chromosome ends (telomeres)

Replication Fork Summary Diagram

                    Helicase
                       ↓
    3'════════════════╗╔════════════════5'
                      ║║   
    5'────────────────╝╚────────────────3'
         Leading strand    Lagging strand
              ↓                  ↓
         Continuous         Discontinuous
         synthesis          (Okazaki fragments)
              ↓                  ↓
    DNA Pol III on both strands
              ↓                  ↓
         One primer         Multiple primers
              ↓                  ↓
    ─────────────────→    ←─── ←─── ←───
                          (fragments joined by ligase)

Accuracy and Proofreading

DNA replication is remarkably accurate: error rate ~1 in 10⁹–10¹⁰ base pairs.

Sources of Accuracy

Proofreading by DNA Polymerase III

1. After adding each nucleotide, polymerase "checks" base pairing

2. Incorrect base causes distortion in DNA structure

3. Polymerase pauses

4. 3'→5' exonuclease activity removes incorrect nucleotide

5. Correct nucleotide added

6. Synthesis continues

Mismatch Repair System

• Operates after replication

• Recognises distortions from mismatched bases

• Distinguishes new strand from template (methylation patterns)

• Removes section containing error

• DNA polymerase fills gap

• Ligase seals

Defects in mismatch repair → increased mutation rate → cancer predisposition (e.g., Lynch syndrome)

Differences Between Prokaryotic and Eukaryotic Replication

Telomeres and the End-Replication Problem

The Problem

Linear chromosomes face a unique challenge:

• RNA primer at the very end of lagging strand cannot be replaced with DNA

• After primer removal, 3' end of template strand has no complement

• Each replication shortens chromosome by ~50–200 bp

This is called the "end-replication problem."

Telomeres

Telomeres are repetitive DNA sequences at chromosome ends that protect against this loss.

Structure:

• Repetitive sequences (in humans: TTAGGG repeated thousands of times)

• Associated proteins (shelterin complex)

• Form protective cap structure

• Do not code for proteins

Function:

• Buffer against shortening

• Protect coding sequences

• Prevent chromosome fusion

• Signal cell age (biological clock)

Telomerase

Telomerase is an enzyme that extends telomeres, counteracting shortening.

Structure:

• Ribonucleoprotein (contains RNA and protein)

• RNA component serves as template for telomere synthesis

• Protein component has reverse transcriptase activity

Mechanism:

1. Telomerase binds to 3' overhang of chromosome

2. Uses internal RNA template to extend 3' end

3. Adds telomeric repeats (TTAGGG in humans)

4. Translocates and repeats

5. Extended strand serves as template for lagging strand synthesis

Expression:

• Active in germ cells, stem cells, and some immune cells

• Inactive in most somatic cells

• Reactivated in ~90% of cancers (allows unlimited division)

Telomeres and Ageing

Hayflick limit:

• Normal somatic cells can only divide ~50–70 times

• After this, telomeres too short → senescence or apoptosis

• Linked to organismal ageing

Progeria:

• Premature ageing disorder

• Accelerated telomere shortening

• Children age rapidly

DNA Replication and the Cell Cycle

S Phase

• DNA replication occurs during S (Synthesis) phase of interphase

• Entire genome replicated once per cell cycle

• Each chromosome replicated → two sister chromatids joined at centromere

• Ensures each daughter cell receives complete genome

Regulation

Licensing system:

• Origins can only fire once per cell cycle

• Pre-replication complexes (pre-RC) assembled in G1

• Licensing factors removed after initiation

• Prevents re-replication

Checkpoints:

• DNA damage checkpoint can halt replication

• Ensures damaged DNA is repaired before cell division

• Defects → genomic instability → cancer

Practical Investigations

Modelling DNA Replication

Physical models:

• Use coloured beads or paper to represent nucleotides

• Show complementary base pairing

• Demonstrate semi-conservative replication

• Show leading and lagging strand synthesis

Analysing Meselson-Stahl Data

Interpreting density gradient results:

• Identify bands by position

• Calculate expected ratios for each generation

• Compare predictions of different models

• Explain why results support semi-conservative model

DNA Extraction

Simple DNA extraction:

1. Blend tissue (e.g., strawberry) with salt/detergent solution

2. Filter to remove debris

3. Add cold ethanol

4. DNA precipitates at interface

5. Spool with glass rod

Demonstrates:

• DNA is a physical molecule

• Can be isolated and studied

Common Exam Questions

Typical Question Types

1. Describe the process of DNA replication (6–8 marks)

   • Helicase unwinds/separates strands

   • Single-strand binding proteins stabilise

   • Primase synthesises RNA primers

   • DNA polymerase III adds nucleotides 5'→3'

   • Leading strand continuous; lagging strand discontinuous (Okazaki fragments)

   • DNA polymerase I removes primers, replaces with DNA

   • DNA ligase joins fragments

2. Explain why replication is semi-conservative (4 marks)

   • Each new molecule has one original strand and one new strand

   • Meselson-Stahl experiment evidence

   • Generation 1: all hybrid (intermediate density)

   • Generation 2: 50% hybrid, 50% light

3. Explain the roles of helicase and DNA polymerase (4 marks)

   • Helicase: unwinds double helix; breaks hydrogen bonds between bases

   • DNA polymerase: adds complementary nucleotides to 3' end

   • Only synthesises in 5'→3' direction

   • Proofreads for accuracy

4. Compare leading and lagging strand synthesis (4 marks)

   • Leading: continuous; one primer; toward fork

   • Lagging: discontinuous; multiple primers; away from fork

   • Both synthesised 5'→3'

   • Lagging requires Okazaki fragment ligation

5. Describe the Meselson-Stahl experiment (5 marks)

   • Bacteria grown in ¹⁵N (heavy)

   • Transferred to ¹⁴N (light)

   • DNA extracted at each generation

   • Separated by density centrifugation

   • Results supported semi-conservative model

6. Explain the end-replication problem and role of telomerase (4 marks)

   • Linear chromosomes cannot replicate very ends

   • Lagging strand primer cannot be replaced

   • Chromosomes shorten with each division

   • Telomerase extends telomeres using RNA template

   • Active in germ cells/stem cells; inactive in somatic cells

Key Terminology Glossary

Summary Comparison Tables

Enzymes in DNA Replication

Leading vs Lagging Strand

Prokaryotic vs Eukaryotic Replication

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