DNA Replication Steps

Why This Matters

DNA replication sits at the heart of two major AP Biology themes: information transfer and cell cycle regulation. When you're asked about how genetic information passes from parent to daughter cells, or why mutations occur despite cellular safeguards, you're really being asked about the molecular machinery of replication. The enzymes and mechanisms here—helicase unwinding, polymerase directionality, ligase sealing—demonstrate how cells solve fundamental problems of copying a double-stranded, antiparallel molecule with incredible accuracy.

This topic connects directly to Unit 6's gene expression concepts (the DNA being copied is the same template used for transcription) and Unit 4's cell cycle checkpoints (S phase is when replication occurs, and the DNA replication checkpoint ensures it's complete before mitosis). On the exam, you won't just be asked to list enzymes—you'll need to explain why the lagging strand requires Okazaki fragments, or how proofreading reduces mutation rates. Don't just memorize the steps; know what problem each enzyme solves and why the process works the way it does.

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Initiation: Setting Up the Replication Fork

Before DNA can be copied, the double helix must be opened and stabilized. These steps create the physical environment where synthesis can occur. The key concept here is that DNA's structure—antiparallel strands held by hydrogen bonds—requires specific enzymes to make it accessible.

DNA Unwinding by Helicase

• Helicase breaks hydrogen bonds between complementary base pairs, separating the two strands at the replication fork
• ATP hydrolysis provides the energy needed to disrupt the A-T and G-C base pairing that holds the helix together
• Creates single-stranded templates—without this step, DNA polymerase cannot access the bases it needs to read

Topoisomerase Action to Relieve Supercoiling

• Topoisomerase cuts and rejoins DNA ahead of the replication fork to release torsional strain caused by unwinding
• Prevents DNA from tangling—as helicase unwinds the helix, the DNA ahead would otherwise become impossibly twisted
• Essential for fork progression—without topoisomerase, replication would stall as supercoiling builds up

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Priming: Getting Synthesis Started

DNA polymerase cannot start synthesis from scratch—it can only add nucleotides to an existing strand. This limitation explains why primase is essential: it creates the $3'$ hydroxyl group that polymerase needs.

Primer Synthesis by Primase

• Primase synthesizes short RNA primers (typically 10-12 nucleotides) complementary to the DNA template
• Provides the $3'$-OH group that DNA polymerase III requires to begin adding nucleotides
• Required for both strands—the leading strand needs one primer, while the lagging strand needs many (one per Okazaki fragment)

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Elongation: Building the New Strands

This is where the actual copying happens, but the antiparallel nature of DNA creates a fundamental asymmetry. DNA polymerase can only synthesize in the $5' \rightarrow 3'$ direction, which means the two strands must be replicated differently.

Leading Strand Synthesis by DNA Polymerase III

• Continuous synthesis occurs because the leading strand runs $3' \rightarrow 5'$, allowing polymerase to follow the replication fork
• Only one primer needed—polymerase III adds nucleotides continuously without interruption
• High speed and efficiency—this strand is replicated faster because there's no need to restart synthesis repeatedly

Lagging Strand Synthesis and Okazaki Fragments

• Discontinuous synthesis is required because the lagging strand runs $5' \rightarrow 3'$, pointing away from the fork
• Okazaki fragments are short DNA segments (100-200 nucleotides in eukaryotes) each initiated by a separate RNA primer
• Multiple primers required—each time the fork advances, a new primer must be laid down to start another fragment

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Maturation: Completing the Daughter Strands

After synthesis, the new strands contain RNA primers and gaps that must be processed. These steps convert a patchwork of fragments into continuous, all-DNA strands.

Removal of RNA Primers

• RNase H degrades RNA primers that were used to initiate synthesis on both strands
• Creates gaps in the sugar-phosphate backbone—these must be filled before the strand is complete
• Critical for DNA integrity—RNA left in the strand would be chemically unstable and prone to degradation

Gap Filling by DNA Polymerase I

• DNA polymerase I replaces RNA with DNA by adding nucleotides to fill the gaps left by primer removal
• $5' \rightarrow 3'$ exonuclease activity allows it to remove primers while simultaneously adding DNA
• Proofreading function ensures that the replacement nucleotides are correctly paired with the template

Nick Sealing by DNA Ligase

• DNA ligase forms phosphodiester bonds between adjacent nucleotides, sealing the sugar-phosphate backbone
• Joins Okazaki fragments into a continuous lagging strand—without ligase, the strand would remain fragmented
• Requires ATP or NAD+ as an energy source to catalyze bond formation

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Quality Control: Ensuring Accuracy

Replication must be extraordinarily accurate—the error rate is approximately 1 in $10^9$ nucleotides. This accuracy comes from multiple layers of proofreading and repair.

Proofreading and Error Correction

• $3' \rightarrow 5'$ exonuclease activity in DNA polymerase allows it to back up and remove mismatched nucleotides
• Immediate correction occurs during synthesis—if the wrong base is added, polymerase detects the distorted helix geometry
• Reduces mutation rate by approximately 100-fold compared to synthesis without proofreading

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End Replication: The Telomere Problem

Linear chromosomes face a unique challenge: the lagging strand cannot be fully replicated at chromosome ends. This "end replication problem" is solved by telomeres and telomerase.

Telomere Replication

• Telomeres are repetitive sequences (TTAGGG in humans) that cap chromosome ends and don't code for proteins
• Telomerase extends telomeres by adding repetitive sequences using its own RNA template—it's a reverse transcriptase
• Active in stem cells and cancer cells—most somatic cells lack telomerase activity, leading to telomere shortening with each division

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Quick Reference Table

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Self-Check Questions

1. Which two enzymes address structural problems caused by the double helix during replication, and how do their functions differ?

2. Why does the lagging strand require multiple RNA primers while the leading strand needs only one? Connect your answer to DNA's antiparallel structure.

3. Compare the roles of DNA Polymerase I and DNA Ligase in completing the lagging strand—what specific problem does each solve?

4. If a cell's proofreading function were disabled, what would happen to the mutation rate, and why does this matter for cell cycle checkpoints?

5. FRQ-style: Explain how the "end replication problem" arises from the mechanism of lagging strand synthesis, and describe how telomerase solves this problem in certain cell types.