DNA Replication Enzymes

Why This Matters

DNA replication sits at the heart of biochemistry's central dogma—without accurate copying of genetic information, cell division fails and life stops. You're being tested on more than enzyme names; exams probe your understanding of how the replication fork functions as a coordinated system, why the leading and lagging strands require different machinery, and what happens when fidelity mechanisms break down. These concepts connect directly to topics like cancer biology, antibiotic mechanisms, and genetic diseases.

Think of the replication fork as a molecular factory where each enzyme has a specialized job. Helicase opens the door, primase lays the foundation, polymerases do the heavy lifting, and ligase cleans up afterward. Don't just memorize what each enzyme does—know where it acts at the fork, why it's necessary, and how its absence would disrupt the entire process. That's what FRQs are really asking.

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Unwinding and Accessing the Template

Before any new DNA can be synthesized, the double helix must be opened and stabilized. These enzymes solve the topological problems inherent in unwinding a twisted, intertwined molecule.

Helicase

• Unwinds the double helix at the replication fork by breaking hydrogen bonds between base pairs—creates the single-stranded templates polymerases need
• Moves 5' to 3' along the lagging strand template, hydrolyzing ATP to power its motor function
• Rate-limiting for fork progression—without helicase, the entire replication complex stalls

Topoisomerase

• Relieves torsional strain ahead of the replication fork by cutting and rejoining DNA strands—prevents the helix from overwinding into positive supercoils
• Type I cuts one strand; Type II (including gyrase) cuts both strands and passes DNA through the gap
• Drug target—fluoroquinolone antibiotics and chemotherapy agents like etoposide inhibit topoisomerases, making this clinically relevant

DNA Gyrase

• Introduces negative supercoils into DNA, counteracting the positive supercoiling caused by helicase—a specialized Type II topoisomerase found in prokaryotes
• ATP-dependent mechanism distinguishes it from relaxing topoisomerases
• Antibiotic target—quinolones specifically inhibit bacterial gyrase, explaining their selectivity for prokaryotes

Single-Strand Binding Proteins (SSB)

• Stabilize single-stranded DNA after helicase unwinds it—prevents reannealing and protects from nuclease degradation
• Bind cooperatively—once one SSB attaches, others bind more readily, coating the entire exposed region
• Essential for polymerase access—keep the template strand extended and readable

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Initiating New Strands

DNA polymerases cannot start synthesis from scratch—they require a pre-existing 3' hydroxyl group. Primase solves this chicken-and-egg problem by synthesizing short RNA primers.

Primase

• Synthesizes RNA primers (typically 10-12 nucleotides) that provide the free 3'-OH group DNA polymerases require
• Acts repeatedly on the lagging strand—each Okazaki fragment needs its own primer, while the leading strand needs only one
• Part of the primosome complex in prokaryotes—associates with helicase for coordinated action at the fork

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Synthesizing and Proofreading New DNA

The polymerases are the workhorses of replication, but they differ dramatically in their roles. Understanding which polymerase does what—and why multiple polymerases exist—is essential for exam success.

DNA Polymerase III

• Primary replicative polymerase in prokaryotes—synthesizes the bulk of new DNA on both leading and lagging strands
• High processivity (adds ~1,000 nucleotides/second) due to the β-clamp sliding clamp that tethers it to DNA
• 3' to 5' exonuclease proofreading reduces error rate to approximately $10^{-7}$—removes misincorporated nucleotides before continuing

DNA Polymerase I

• Removes RNA primers using its unique 5' to 3' exonuclease activity—no other replicative polymerase has this capability
• Fills gaps left by primer removal with DNA nucleotides, working in the standard 5' to 3' direction
• Lower processivity than Pol III—suited for short gap-filling rather than bulk synthesis

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Removing Primers and Joining Fragments

The lagging strand is synthesized as discontinuous Okazaki fragments, each initiated by an RNA primer. These enzymes convert a patchwork of RNA-DNA segments into a continuous DNA strand.

RNase H

• Degrades RNA in RNA-DNA hybrids—specifically recognizes and cleaves the RNA portion of the primer-template junction
• Works upstream of Pol I—creates the gap that Pol I then fills with DNA
• Cannot remove the final ribonucleotide directly bonded to DNA—this requires additional processing

Ligase

• Seals phosphodiester backbone nicks by catalyzing bond formation between adjacent 3'-OH and 5'-phosphate groups
• Requires energy cofactor—ATP in eukaryotes and bacteriophages; NAD⁺ in bacteria
• Final step in Okazaki fragment maturation—without ligase, the lagging strand remains fragmented and non-functional

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Maintaining Chromosome Ends

Linear chromosomes face the "end replication problem"—conventional polymerases cannot fully replicate chromosome termini. Telomerase provides a specialized solution unique to eukaryotes.

Telomerase

• Extends telomeres by adding repetitive sequences (TTAGGG in humans) to chromosome 3' ends—compensates for the sequences lost each replication cycle
• Carries its own RNA template—functions as a reverse transcriptase, synthesizing DNA from an RNA template it contains
• Highly active in stem cells and cancer cells—reactivation of telomerase is a hallmark of ~90% of cancers, making it a therapeutic target

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Quick Reference Table

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Self-Check Questions

1. Which two enzymes both address problems caused by unwinding the double helix, and how do their mechanisms differ?

2. A mutation eliminates all 5' to 3' exonuclease activity in a cell. Which enzyme is affected, and what specific defect would you observe at the replication fork?

3. Compare the roles of DNA Polymerase I and DNA Polymerase III—why does the cell need both, and what would happen if you only had Pol III?

4. An FRQ asks you to explain why the lagging strand requires more enzymatic steps than the leading strand. Which enzymes would you discuss, and in what order do they act?

5. Telomerase and primase both synthesize nucleic acids that serve as substrates for DNA polymerase. What is fundamentally different about what each enzyme produces and why?