Essay plan - accuracy and fidelity of replication and segregation of the genome

What is meant by fidelity in DNA replication?

Fidelity refers to replicated DNA strands being exact copies of their template strands, with minimal mutations.

What is meant by accuracy in DNA replication and segregation?

Accuracy refers to ensuring each daughter cell receives the correct number and identity of chromosomes.

Why are replication and segregation particularly challenging in eukaryotes?

Eukaryotes have large genomes with multiple origins of replication, which must be replicated once and only once and then correctly segregated.

What are the consequences of replication or segregation failure?

Gene dosage imbalance, mutations, genome instability, aneuploidy (e.g. trisomy 21).

Which polymerases replicate the leading and lagging strands in eukaryotes?

Pol ε synthesises the leading strand; pol δ synthesises the lagging strand.

What makes replicative DNA polymerases “high fidelity”?

They selectively incorporate correct nucleotides, exclude ribonucleotides, and proofread errors using exonuclease activity.

What is steric gating in DNA polymerases?

A mechanism where ribonucleotides are excluded from the active site due to steric clashes with active-site residues.

How do DNA polymerases sense correct base pairing?

The palm domain senses helix geometry formed by correct Watson–Crick base pairing.

What happens when an incorrect nucleotide is incorporated?

Helix geometry is distorted, slowing synthesis and preventing correct alignment for nucleophilic attack.

How does proofreading by DNA polymerases work?

Misincorporation destabilises the primer-template junction, shifting it from the polymerase site to the 3′–5′ exonuclease site where the incorrect nucleotide is removed.

Why is proofreading kinetically favoured over mismatch extension?

he rate of exonuclease excision is higher than the rate of extension from a mismatched base.

What structural insight explains how polymerases reject mismatches?

Structures show an “ajar” conformation when mismatches are present, preventing efficient catalysis.

What is origin licensing?

The loading of inactive MCM2–7 helicase complexes onto DNA to form pre-replication complexes (pre-RCs).

When does origin licensing occur in the cell cycle?

Late M phase and G1 phase.

Why must licensing and firing be temporally separated?

To ensure the genome is replicated once and only once per cell cycle.

Which proteins are required for origin licensing?

ORC, Cdc6, and Cdt1.

How does geminin regulate origin licensing?

Geminin binds and inhibits Cdt1, preventing MCM loading.

How is geminin itself regulated?

It is degraded by APC/C during M and G1, allowing licensing, and accumulates in S phase to prevent re-licensing.

How do S-phase CDKs inhibit origin licensing?

By phosphorylating licensing components, targeting them for degradation, nuclear export, or preventing protein interactions.

Give examples of CDK-mediated inhibition of licensing.

• Phosphorylation of Cdc6 → SCF-mediated degradation

• Phosphorylation of ORC → inhibits Cdt1 binding

• Phosphorylation of MCM → nuclear export

How is Cdt1 degraded during S phase independently of CDKs?

Through a Cul4–Ddb1–Rbx1 E3 ubiquitin ligase when Cdt1 binds PCNA.

What is origin firing?

Activation of the MCM helicase to form the CMG complex and initiate DNA unwinding.

Which kinases trigger origin firing?

S-CDK and DDK.

What is the CMG complex?

Cdc45–MCM–GINS, the active replicative helicase.

How does S-CDK promote CMG assembly?

By phosphorylating Sld2 and Sld3, creating binding sites for Dpb11 and recruiting Pol ε and GINS.

What role does DDK play in firing?

Phosphorylates MCM N-termini to promote interaction with Pol ε and enhance firing.

How does firing inhibit re-licensing?

CDK activity phosphorylates unloaded MCMs, causing nuclear export and preventing new licensing.

Why must replication termination be tightly regulated?

Premature helicase removal could leave regions unreplicated.

How are active and terminated helicases distinguished?

Active helicases bind ssDNA; terminated helicases bind dsDNA when forks converge.

What triggers CMG removal during termination?

Binding to dsDNA creates a site for Dia2-mediated ubiquitinylation.

What removes ubiquitinylated CMG from DNA?

The ATPase p97.

Are terminated CMG complexes degraded?

No, they are removed from DNA and recycled.

Why must Okazaki RNA primers be removed?

RNA is error-prone and must not remain in DNA.

Why does decatenation need to occur after replication?

Sister chromatids are topologically interlinked and must be separated for segregation.

Which enzyme performs decatenation?

Topoisomerase II.

What is the risk if decatenation fails?

Chromosome breakage or failure of sister chromatid segregation.

What is the purpose of mismatch repair?

To correct replication errors that escape polymerase proofreading.

What is the general mechanism of mismatch repair?

Mismatch recognition → incision → excision → resynthesis → ligation.

How do prokaryotes identify the newly synthesised strand?

By recognising hemi-methylated DNA.

Why is mismatch repair considered a “second line of defence”?

It corrects errors missed by DNA polymerase proofreading.

Why must chromosomes be condensed before segregation?

Decondensed DNA would tangle and break during segregation.

What is condensin?

An SMC ATPase complex that compacts chromosomes.

How does condensin compact chromosomes?

Through loop extrusion, forming a condensed chromosome axis.

What happens when condensin I and II are knocked out?

Cells fail to form mitotic chromosomes and cannot segregate DNA, leading to cell death

What problem does cohesin solve?

Maintaining sister chromatid identity until anaphase.

What is the ring model of cohesin?

Cohesin forms a ring that topologically entraps sister chromatids.

How do cross-linking experiments support the ring model?

Covalently locking cohesin subunits does not disrupt cohesion, indicating cohesion is not protein–DNA binding but topological entrapment.

Why is cohesin dynamic in G1?

Wpl–Pds5 stimulates ATPase activity, opening the ring and allowing loading/unloading.

How is stable cohesion established during S phase?

Smc3 acetylation by Eco acetyltransferases inhibits Wpl–Pds5 binding

Why is Smc3 acetylation S-phase specific?

Eco acetyltransferases are only active during S phase.

How do vertebrates further stabilise cohesin?

Sororin binds Pds5, preventing Wpl-mediated ring opening.

Why does sororin only act in S phase?

It is degraded by APC/C outside S phase.

What enzyme cleaves cohesin at anaphase?

Separase.

How is separase kept inactive before anaphase?

It is bound by securin.

Why is this control by SAC critical?

Ensures sister chromatids separate only when properly attached, preventing mis-segregation.

How does the spindle assembly checkpoint (SAC) regulate anaphase?

Unattached kinetochores form the MCC, which inhibits APC/C

What triggers anaphase onset?

All kinetochores attach → APC/C ubiquitinylates securin → separase is activated.