Enzymes in DNA Replication
This is the process of creating two identical DNA copies from one original molecule. Key enzymes act in a coordinated manner at the replication fork.
- Helicase:
- Function: Unwinds the DNA double helix at the origin of replication by breaking hydrogen bonds, creating the replication fork. This process requires ATP.
- Prokaryotes vs. Eukaryotes: In prokaryotes, DnaB is the primary helicase. In eukaryotes, the MCM2-7 complex functions as the helicase.
- Topoisomerases (e.g., DNA Gyrase):
- Function: Relieve the torsional stress and supercoiling that builds up ahead of the replication fork as DNA unwinds. They do this by creating temporary nicks in the DNA backbone.
- Clinical Correlation: Fluoroquinolones (e.g., ciprofloxacin) are antibiotics that inhibit prokaryotic topoisomerase II (DNA gyrase) and topoisomerase IV. Etoposide is a chemotherapy agent that inhibits eukaryotic topoisomerase II.
- Primase:
- Function: Synthesizes a short RNA primer (5-10 nucleotides) on the DNA template. This is crucial because DNA polymerase cannot start synthesis de novo and requires a free 3’-OH group to begin adding nucleotides.
- Prokaryotes vs. Eukaryotes: In prokaryotes, this is done by DnaG. In eukaryotes, primase activity is part of DNA Polymerase α.
- DNA Polymerase:
- Function: The main enzyme for synthesizing new DNA. It reads the template strand in the 3’→5’ direction and synthesizes the new strand in the 5’→3’ direction. Many polymerases also have a 3’→5’ exonuclease (“proofreading”) activity to correct errors.
- Prokaryotes: Have DNA Polymerase I, II, and III. DNA Pol III is the primary enzyme for elongation. DNA Pol I has 5’→3’ exonuclease activity to remove RNA primers and replaces them with DNA.
- Eukaryotes: Have multiple DNA polymerases. Pol δ synthesizes the lagging strand, Pol ε synthesizes the leading strand, and Pol α contains primase activity and starts DNA synthesis.
- DNA Ligase:
- Function: Joins DNA fragments together by forming a phosphodiester bond. It is essential for connecting the Okazaki fragments on the lagging strand and sealing final nicks in the DNA backbone.
- Telomerase:
- Function: A reverse transcriptase enzyme found only in eukaryotes that adds repetitive TTAGGG sequences to the 3’ ends of chromosomes (telomeres). This prevents the loss of genetic material from the ends of linear chromosomes with each round of replication (addressing the “end-replication problem”).
Enzymes in Transcription
This is the synthesis of an RNA strand from a DNA template.
- RNA Polymerase:
- Function: The primary enzyme responsible for transcription. It unwinds a portion of the DNA helix, reads the template strand (antisense strand), and synthesizes a complementary RNA strand in the 5’→3’ direction. Unlike DNA polymerase, it does not require a primer to initiate synthesis.
- Prokaryotes: A single RNA polymerase synthesizes all types of RNA (mRNA, tRNA, rRNA). It requires a sigma (σ) factor to recognize the promoter region and initiate transcription.
- Eukaryotes: Have three distinct RNA polymerases:
- RNA Polymerase I: Located in the nucleolus; synthesizes most rRNA.
- RNA Polymerase II: Located in the nucleoplasm; synthesizes mRNA, snRNA (small nuclear RNA), and microRNA. It requires transcription factors to bind to promoter regions like the TATA box.
- RNA Polymerase III: Located in the nucleoplasm; synthesizes tRNA and 5S rRNA.
- Clinical Correlation:
- α-amanitin, found in Amanita phalloides (death cap mushrooms), is a potent inhibitor of RNA Polymerase II, causing severe liver toxicity.
- Rifampin is an antibiotic that inhibits prokaryotic RNA polymerase.
- Transcription Factors (Eukaryotes):
- Function: A group of proteins that bind to specific DNA sequences (promoters, enhancers) to help recruit and position RNA polymerase II correctly at the transcription start site, thereby regulating gene expression. They are essential for the initiation of transcription in eukaryotes.
| Synthesizing Polymerase | Type of RNA Produced | Function |
|---|---|---|
| RNA polymerase I | 18S, 5.8S & 28S ribosomal RNA | Forms essential ribosomal components |
| RNA polymerase II | mRNA | Translated by ribosomes to form specific proteins |
| Small nuclear RNA | Involved in mRNA splicing & transcription regulation | |
| MicroRNA | Causes gene silencing via translation arrest or mRNA degradation | |
| RNA polymerase III | Transfer RNA | Adaptor molecule linking codons with specific amino acids |
| 5S ribosomal RNA | Essential component of 60S ribosomal subunit |
Mnemonic
I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA.
A fundamental concept in molecular biology is that synthesis of new DNA and RNA strands always occurs in the 5’ → 3’ direction. This means nucleotides are added to the 3’-hydroxyl (-OH) group of the growing strand. However, the enzymes read the template strand in the opposite direction.
5’ → 3’ Processes/Enzymes
This is the direction of synthesis for all new nucleic acid chains.
- DNA Polymerase (Replication)
- 5’ → 3’ Polymerase Activity: Synthesizes the new DNA strand by adding dNTPs to the 3’ end. This is true for both the leading and lagging strands (Okazaki fragments).
- 5’ → 3’ Exonuclease Activity: Found in DNA Polymerase I (prokaryotes). This activity is crucial for removing the RNA primers from the lagging strand and replacing them with DNA.
- RNA Polymerase (Transcription)
- 5’ → 3’ Polymerase Activity: Synthesizes the new mRNA strand using the DNA template. It adds ribonucleotides to the 3’ end of the growing RNA molecule.
- Ribosome (Translation)
- The ribosome reads the mRNA transcript in the 5’ → 3’ direction to synthesize protein from the N-terminus to the C-terminus.
3’ → 5’ Processes/Enzymes
This directionality is associated with reading the template strand and proofreading.
- Template Strand Reading
- DNA Polymerase (Replication): Moves along the template strand in the 3’ → 5’ direction to synthesize the new 5’ → 3’ strand.
- RNA Polymerase (Transcription): Reads the DNA template (antisense) strand in the 3’ → 5’ direction to create the complementary 5’ → 3’ mRNA transcript.
- Exonuclease (Proofreading) Activity
- 3’ → 5’ Exonuclease Activity: This is the proofreading function found in high-fidelity DNA polymerases (like DNA Pol I, II, and III in prokaryotes). If an incorrect nucleotide is added, the polymerase can back up and excise the mismatched base from the 3’ end of the new strand before continuing synthesis. This significantly increases replication fidelity.
| Process/Enzyme | Activity | Direction | Function |
|---|---|---|---|
| DNA & RNA Polymerase | Synthesis (Polymerase) | 5’ → 3’ | Elongating the new nucleic acid strand |
| DNA Polymerase | Reading Template Strand | 3’ → 5’ | Guiding the synthesis of the new strand |
| RNA Polymerase | Reading Template Strand | 3’ → 5’ | Guiding the synthesis of mRNA |
| DNA Polymerase I | Exonuclease | 5’ → 3’ | Removing RNA primers |
| DNA Polymerase (most) | Exonuclease (Proofreading) | 3’ → 5’ | Excising mismatched nucleotides |
Exonuclease vs. Endonuclease
| Feature | Exonuclease | Endonuclease |
|---|---|---|
| Action | Removes nucleotides from the ends (5’ or 3’) of a nucleic acid chain. | Cleaves phosphodiester bonds within a nucleic acid chain. |
| Products | Single mononucleotides, released one at a time. | Oligonucleotide fragments of varying sizes. |
| Specificity | Generally not sequence-specific. | Can be non-specific (e.g., DNase I) or highly sequence-specific (e.g., restriction enzymes). |
| Key Example | DNA Polymerase Proofreading: 3’→5’ exonuclease activity removes mismatched bases during replication. | Restriction Enzymes: Recognize and cut specific palindromic DNA sequences (e.g., for RFLP, cloning). |
| Circular DNA | Cannot act on circular DNA (no free ends). | Can act on circular DNA. |