DNA Replication Process

Why This Matters

DNA replication sits at the heart of cellular reproduction and genetic inheritance—two themes that connect Unit 4's cell cycle regulation to Unit 6's gene expression and even Unit 7's discussions of mutation and evolution. When you're tested on replication, you're really being tested on your understanding of enzyme specificity, directionality constraints, energy requirements, and error correction mechanisms. The College Board loves asking how cells maintain genetic fidelity across generations, and replication is where that story begins.

Think of replication as a coordinated molecular assembly line where every enzyme has a specific job dictated by the chemistry of DNA itself. The 5' to 3' directionality constraint explains why we have leading and lagging strands. The need for a free 3'-OH group explains why primers exist. Don't just memorize the enzyme names—know why each step is necessary and how errors at any stage could lead to mutations that drive evolution or disease.

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The Foundation: Replication Model and Starting Points

Before any enzymes get to work, cells must solve two problems: where does replication start, and how do we preserve genetic information? The semi-conservative model and origins of replication answer these fundamental questions.

Semi-Conservative Replication Model

• Each daughter molecule contains one parental strand and one new strand—this ensures genetic continuity while allowing efficient copying
• Meselson-Stahl experiment confirmed this model using $^{15}N$ and $^{14}N$ isotopes to track DNA density across generations
• Preserves template information for error-checking, since the original strand serves as a reference during proofreading

Origin of Replication

• Specific DNA sequences where initiator proteins bind and recruit the replication machinery—these AT-rich regions unwind more easily due to fewer hydrogen bonds
• Eukaryotes have multiple origins per chromosome, allowing simultaneous replication from many sites to copy large genomes quickly
• Prokaryotes typically have one origin (oriC), creating a single replication bubble that proceeds bidirectionally

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Unwinding and Access: Creating the Template

DNA's double helix must be opened before copying can begin. This section covers how cells separate the strands and maintain access to the template.

DNA Helicase and Unwinding of the Double Helix

• Breaks hydrogen bonds between complementary bases—uses ATP hydrolysis to power the unwinding of the double helix
• Creates single-stranded templates that DNA polymerase can read and copy
• Works at the replication fork, the Y-shaped junction where active synthesis occurs

Replication Fork

• Y-shaped structure where the parental DNA separates and both new strands are synthesized simultaneously
• Moves bidirectionally from each origin, with helicase continuously unwinding ahead of the polymerases
• Coordinates multiple enzymes—helicase, primase, polymerase, and ligase all work together at this dynamic site

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Building New Strands: The Directionality Problem

DNA polymerase can only add nucleotides in one direction, creating an asymmetry problem at the replication fork. This constraint explains why leading and lagging strand synthesis differ so dramatically.

DNA Polymerase and Its Role in Nucleotide Addition

• Adds nucleotides only to the 3' end—requires a pre-existing strand with a free 3'-OH group, which is why primers are essential
• Catalyzes phosphodiester bond formation between the 3'-OH of the growing strand and the 5'-phosphate of the incoming nucleotide
• Has 3' to 5' exonuclease activity for proofreading, allowing it to remove and replace mismatched bases

Primase and RNA Primers

• Primase synthesizes short RNA primers (~10 nucleotides) that provide the free 3'-OH group DNA polymerase requires
• No template-independent synthesis is possible for DNA polymerase—this is a fundamental limitation of the enzyme's active site
• Primers are later removed and replaced with DNA by a different polymerase, then sealed by ligase

Leading Strand Synthesis

• Synthesized continuously in the 5' to 3' direction toward the replication fork as helicase unwinds ahead
• Requires only one RNA primer to initiate, then polymerase adds nucleotides without interruption
• Faster and simpler than lagging strand synthesis because the direction of synthesis matches fork movement

Lagging Strand Synthesis and Okazaki Fragments

• Synthesized discontinuously as short fragments (100-200 nucleotides in eukaryotes) because polymerase must work away from the fork
• Each Okazaki fragment requires its own RNA primer—primase must repeatedly initiate synthesis as new template is exposed
• Fragments are later joined after primer removal, making this strand more complex and error-prone to synthesize

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Finishing the Job: Sealing and Proofreading

Replication isn't complete until fragments are joined and errors are corrected. These final steps ensure the integrity of the genetic information being passed to daughter cells.

DNA Ligase and Joining DNA Fragments

• Forms phosphodiester bonds between the 3'-OH of one fragment and the 5'-phosphate of the next, sealing the sugar-phosphate backbone
• Essential for lagging strand completion—without ligase, Okazaki fragments would remain disconnected
• Requires ATP or NAD+ as an energy source to catalyze bond formation

Proofreading and Error Correction Mechanisms

• DNA polymerase proofreads during synthesis—its 3' to 5' exonuclease activity removes mismatched nucleotides immediately
• Error rate drops from ~1 in $10^5$ to ~1 in $10^7$ after proofreading, with mismatch repair reducing it further to ~1 in $10^9$
• Uncorrected errors become mutations—connecting replication fidelity to genetic variation and evolution (Topic 7.4)

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Quick Reference Table

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Self-Check Questions

1. Which two enzymes both require energy (ATP) to perform their functions during replication, and what does each use that energy for?

2. Explain why the lagging strand requires multiple RNA primers while the leading strand needs only one. What structural feature of DNA creates this difference?

3. Compare the roles of DNA polymerase's proofreading function and DNA ligase—how do their molecular actions differ, and at what stage of replication does each operate?

4. If a mutation inactivated primase, which strand(s) would be affected, and why can't DNA polymerase simply start synthesis without primers?

5. An FRQ asks you to explain how DNA replication maintains genetic fidelity. Identify three mechanisms discussed in this guide and explain how each reduces the error rate.