DNA replication

semi-conservative replication

• Each daughter duplex = 1 parental + 1 nascent strand

• Ensures error-checking via template complementarity

Chemical constraint of DNA synthesis

DNA polymerases:

• Require:

  • Template strand

  • Primer with free 3′-OH

• Catalyse:

  • Nucleophilic attack of 3′-OH on α-phosphate of incoming dNTP

Releases pyrophosphate (PPi) → drives reaction forward

Antiparallel nature creates asymmetry

Because strands are antiparallel:

• Leading strand: continuous synthesis

• Lagging strand: discontinuous synthesis

This is a fundamental structural constraint, not a regulatory choice

Replication Fork

The replisome is including:

• CMG helicase

• DNA polymerases (α, δ, ε)

• PCNA (sliding clamp)

• RFC (clamp loader)

• RPA (ssDNA binding protein)

• Topoisomerases

Initiation of replication

• Origin Licensing (G1 Phase)

• Helicase Activation (S Phase)

Origin Licensing (G1 Phase)

goal is to Load helicase in an inactive form

Step 1: ORC binding

• ORC binds double-stranded DNA at origins

• Acts as a platform for assembly

Step 2: Recruitment of licensing factors

• Cdc6 (ATPase)

• Cdt1

Step 3: Loading of MCM2–7 helicase

• Two MCM hexamers loaded:

  • Form double hexamer

  • Arranged head-to-head

MCM encircles double-stranded DNA

Not yet unwinding → topologically loaded

Licensing is restricted to G1 because Licensing proteins are later degraded/inactivated

Helicase Activation (S Phase)

• triggered by Activation of DDK and S-CDK cascades

• irreversible within the cell cycle

• tightly linked to CDK activity → ensures once-per-cycle firing

DDK cascade

Phosphorylates MCM → enables:

• Recruitment of Cdc45

• Recruitment of Sld3

S-CDK cascade

• Phosphorylates: Sld2 and Sld3

→ allows binding of:

• Dpb11

• GINS complex

• DNA polymerase ε

formation of active helicase

CMG complex = Cdc45 + MCM + GINS

structural transition in helicase activation

• MCM:

  • From dsDNA-bound → ssDNA-translocating

• DNA strands separated

• Replication bubble forms

elongation phase

• primer synthesis

• Processive DNA Synthesis

primer synthesis in elongation phase

Performed by DNA polymerase α–primase complex

Mechanism:

1. Primase synthesizes RNA (~10 nt)

2. Pol α extends with DNA (~20–30 nt)

• Pol α has no proofreading

• Low fidelity → replaced quickly

Processive DNA Synthesis in elongation phase

• Sliding clamp system:

  • PCNA (Proliferating Cell Nuclear Antigen)

  • Clamp Loader (RFC)

• leading strand synthesis

• lagging strand synthesis

PCNA (Proliferating Cell Nuclear Antigen) in sliding clamp system

• Ring-shaped protein

• Encircles DNA

• Tethers polymerase → increases processivity

Clamp Loader (RFC) in sliding clamp system

• Uses ATP

• Loads PCNA onto DNA

leading strand synthesis

• Enzyme: DNA polymerase ε

• Continuous synthesis

• Coupled directly to helicase

lagging strand synthesis

• repetitive and discontinuous

• tightly synchronised with leading strand

• Okazaki Fragment Cycle

• Strand Looping Model

Okazaki Fragment Cycle

Each fragment involves:

1. Primer synthesis (Pol α)

2. Extension (Pol δ)

3. Strand displacement

4. Primer removal

5. Ligation

strand looping model

• Lagging strand forms loop

• Allows both polymerases to move with fork

Maintains coordination

Catalytic mechanism of DNA polymerases

• Requires 2 Mg²⁺ ions

• Mechanism:

  • One activates 3′-OH

  • One stabilises negative charge

Proofreading Mechanism of DNA polymerases

• Incorrect base:

  • Causes distortion

  • DNA shifts to exonuclease site

• Base removed → synthesis resumes

Pol α

low processivity

Pol δ / ε

High (with PCNA)

Pol α → Pol δ/ε transition is tightly regulated

MCM Helicase Properties

• Hexameric ring structure

• ATP-dependent motor

DNA helices mechanism

• Encircles one strand (lagging strand template)

• Moves 3′ → 5′

• Forces strand separation

• RPA binds ssDNA

• Prevents:

  • Reannealing

  • Secondary structures

• Helicase activity is physically coupled to:

  • Polymerase activity

  • Chromatin disruption

DNA Topoisomerases

Supercoiling problem: Helicase unwinding generates Positive supercoils ahead of fork

Topoisomerases prevent:

• Fork stalling

• DNA breakage

• Replication collapse

Type I Topoisomerase

• Cuts one strand

• Forms transient covalent bond

• Allows rotation

Type II Topoisomerase

• Cuts both strands

• Passes another duplex through break

• Reseals DNA

Regulation of Replication

• Core principle: Origins must fire once and only once

• Liscencing control (G1 only) - MCM loading permitted

• Firing control (S phase only) - Requires CDK + DDK

Prevention of Re-Replication in metazoans

• Cdt1 degradation

• Cdc6 degradation

• Geminin binds Cdt1

• ORC phosphorylation

Geminin Mechanism in Prevention of Re-Replication

• Present in:

  • S phase

  • G2

  • M phase

• Inhibits Cdt1 → prevents MCM loading

APC (Anaphase Promoting Complex)

Degrades Geminin at mitosis
→ allows next G1 licensing

Origin Efficiency & Stochastic Firing

Not all licensed origins fire

Late replication ≠ late firing
May reflect inefficiency

Influencing Factors:

• Limiting initiation proteins

• Chromatin environment

• Rif1-mediated inhibition

Passive Replication

• If origin doesn’t fire:
  → replicated by neighbouring fork

End-Replication Problem

DNA polymerase cannot:

• Initiate synthesis de novo

• Replace final RNA primer

Consequences

• Progressive telomere shortening

• Loss of genetic material over time

Additional Mechanism:

• Fork disassembly before complete synthesis

Telomerase

Enzyme Type: Reverse transcriptase

Structure:

• TERT (protein)

• RNA template

Telomerase catalytic cycle

• Binding to 3′ overhang

• Base pairing with RNA template

• DNA extension

• Translocation

• Repeat synthesis

can add hundreds of repeats

regulation of telomere length

• Only subset of telomeres elongated

• Balance between:

  • shortening

  • elongation

Telomere length = dynamic equilibrium

Chromatin Replication

Problem:

DNA is tightly packed in nucleosomes

Step 1. Nucleosome disruption

• CMG helicase + FACT

Step 2. Histone recycling

• Old H3/H4 tetramers redistributed

Step 3. New histone deposition

• CAF1 + ASF1

Epigenetic Inheritance

• Old histones retain modifications

• Guide modification of new histones

Replication preserves:

• DNA sequence

• Chromatin state

Initiation: How DNA Unwinding Begins at Origins of Replication

Replication begins at specific DNA sequences called origins of replication (ori), where DNA is locally unwound.

Initiation in bacteria (e.g. E.coli)

• Origin recognition

• DNA unwinding

• Helicase loading

• Stabilisation of single strands

• Relieving supercoiling

• Initiation is tightly controlled (only once per cell cycle).

• Formation of replication forks marks the true start of DNA synthesis

1. Origin recognition

• The origin (oriC) contains repeated sequences.

• The initiator protein DnaA binds to these repeats.

• ATP-bound DnaA oligomerizes → wraps DNA → introduces strain.

2. DNA unwinding

• AT-rich region adjacent to DnaA binding sites melts (easier to separate due to fewer H-bonds).

• This creates an open complex.

3. Helicase loading

• Helicase = DnaB

• Loaded onto DNA by DnaC (helicase loader).

• DnaB begins unwinding DNA using ATP → creates replication forks.

4. Stabilisation of single strands

• SSB proteins (single-strand binding proteins) bind exposed DNA.

• Prevent re-annealing and secondary structures.

5. Relieving supercoiling

Topoisomerase (DNA gyrase) relieves torsional stress ahead of fork.

RNA Primer Synthesis by DNA Primase

DNA polymerases cannot start synthesis de novo → require a primer

• Primase is part of the primosome (with helicase).

• Required:

  • Once on leading strand

  • Repeatedly on lagging strand

RNA Primer Synthesis by DNA Primase mechanism

• Enzyme: Primase (DnaG in bacteria)

Steps:

1. Primase binds to single-stranded DNA at the replication fork.

2. Synthesizes a short RNA primer (~10–12 nucleotides):

   • Uses ribonucleotides (ATP, GTP, etc.)

   • Synthesizes 5′ → 3′

3. Primer provides a free 3′-OH group.

Loading the Sliding Clamp

The sliding clamp ensures high processivity of DNA polymerase

• Sliding clamp: β-clamp (bacteria)

• Clamp loader complex: uses ATP

• Without clamp → polymerase falls off frequently.

• With clamp → can synthesize thousands of nucleotides continuously

Mechanism of loading the sliding clamp

• Clamp loader binds ATP → changes conformation.

• Opens the β-clamp ring.

• Positions clamp around DNA at primer-template junction.

• ATP hydrolysis:

  • Releases clamp loader

  • Leaves clamp locked onto DNA

• DNA polymerase binds clamp → becomes highly processive.

Coordination of Leading and Lagging Strand Synthesis

problem: DNA is antiparallel, but polymerase only works 5′ → 3′.

solution: Semi-discontinuous replication

Key proteins:

• DNA polymerase III (main replicative enzyme)

• Sliding clamp

• Clamp loader

• Primase

• Helicase

Prevents exposure of large amounts of ssDNA

Ensures both strands replicate at same rate

Semi-discontinuous replication in leading strand

• Synthesized continuously

• Same direction as replication fork movement

Semi-discontinuous replication in lagging strand

• Synthesized discontinuously

• Forms Okazaki fragments

Replisome Coordination Model

Key concept: Trombone model

• Lagging strand loops out so both polymerases move in same direction.

Replisome Coordination Model mechanism

• Helicase unwinds DNA.

• Leading strand polymerase synthesizes continuously.

• Lagging strand:

  • Primase lays RNA primer

  • DNA polymerase synthesizes fragment

• Loop grows (like trombone slide).

• When fragment completed:

  • Polymerase releases

  • New clamp loaded

  • New primer synthesized

  • Loop resets

Maturation of Okazaki Fragments

Problem: Lagging strand fragments contain RNA primers → must be removed and replaced

Mechanism:

1. Primer removal

• Enzyme: DNA polymerase I

• Has 5′ → 3′ exonuclease activity

• Removes RNA primer while adding DNA

2. Gap filling

• DNA polymerase I replaces RNA with DNA

3. Nick sealing

• Enzyme: DNA ligase

Reaction:

• Forms phosphodiester bond between fragments

• Uses:

  • ATP (eukaryotes)

  • NAD⁺ (bacteria)

Final result:

• Continuous lagging strand with no breaks

Termination of DNA Replication in Bacteria

Replication ends when forks meet at termination sites

Termination of DNA Replication in Bacteria in E. coli

Ter sites:

• Specific DNA sequences opposite origin

Tus protein:

• Binds Ter sites → blocks helicase

Mechanism:

1. Replication forks move bidirectionally from origin.

2. Tus-Ter complex allows fork entry from one direction only.

3. Forks meet → replication stops.

Final steps:

1. Removal of remaining primers

• DNA polymerase I + ligase

2. Separation of daughter chromosomes

• Problem: chromosomes may be interlinked (catenanes)

Solution: Topoisomerase IV - Cuts and separates DNA molecules

Outcome: Two fully replicated, separate circular chromosomes

Chromatin Context (Eukaryotes)

Even though your objectives focus on replication, your textbook adds important context:

• DNA is packaged into chromatin

• Replication must:

  • Disrupt nucleosomes

  • Reassemble them afterward

Epigenetic inheritance:

• Histone modifications and structures (e.g. centromeres) are re-established after replication

Telomeres and End-Replication Problem

Problem:

• Linear chromosomes lose DNA at ends after replication

Solution:

• Telomerase

  • Extends 3′ end using RNA template

  • Prevents chromosome shortening

Genome Stability

Replication must integrate with:

• Centromere function (accurate segregation)

• Chromatin domains

• Boundary elements (prevent inappropriate gene silencing)

he End-Replication Problem at Telomeres

DNA replication cannot fully copy the ends of linear chromosomes, leading to progressive shortening.

1. primer dependence

2. Final Primer Removal Problem

3. Fork Dynamics Contribution

primer dependence

• DNA polymerases require an RNA primer.

• On the lagging strand, synthesis occurs in Okazaki fragments.

Final Primer Removal Problem

• The last RNA primer at the 5′ end:

  • Is removed

  • Leaves a gap

  • Cannot be filled → no upstream 3′-OH

Result: Loss of terminal DNA sequence

Fork Dynamics Contribution

• Replication fork may disassemble:

  • Leading strand may finish first

  • Lagging strand incomplete

• Can result in loss of terminal Okazaki fragment

consequences of progressive shortening

• Chromosomes shorten every cell division

• Leads to:

  • Genomic instability

  • Activation of DNA damage response

  • Cellular senescence

How telomeres solving the progressive shortening

• Telomeres consist of repetitive DNA:

  • Humans: TTAGGG repeats

• Features:

  • G-rich strand (3′ overhang)

  • Length: ~50 bp → >30,000 bp

• Form structures:

  • t-loops (protect chromosome ends)

Telomeres:

• Distinguish chromosome ends from DNA breaks

• Prevent:

  • DNA repair activation

  • End-to-end chromosome fusion

What is Telomerase

A specialised reverse transcriptase:

• Extends telomeres using an RNA template

components of telomerase

1. Protein Component

• TERT (telomerase reverse transcriptase)

• Catalyses DNA synthesis

2. RNA Component

• Provides template sequence

• Determines repeat sequence

telomeramase mechanism

Step 1: Binding

• Telomerase binds to:

  • 3′ G-rich overhang

---

Step 2: Base Pairing

• DNA 3′ end pairs with:

  • RNA template within telomerase

---

Step 3: Extension

• TERT synthesises DNA:

  • Adds telomeric repeats (5′ → 3′)

---

Step 4: Translocation

• Enzyme shifts forward

• Repeats synthesis

➡ Leads to progressive elongation

---

Step 5: Lagging Strand Completion

• DNA pol α:

  • Synthesises RNA primer

  • Fills in complementary C-rich strand

---

📍 Key Details

• Telomerase extends only the G-rich strand

• The C-strand is filled later