DNA Replication - Notes

Key Definition

Main Components Involved (Table 24.1)

REPLICATION IN PROKARYOTES

1. Semiconservative Replication

• Parent DNA has two complementary strands; both undergo simultaneous replication
• Each daughter molecule has one original strand + one new strand
• This is called semiconservative replication - half of original DNA is conserved
• First experimental evidence: Meselson and Stahl (1958)

2. Initiation of Replication

• DNA synthesis begins at a site called the origin of replication
• In prokaryotes: single origin of replication
• Sites mostly consist of short A-T base pair sequences
• Protein dna A (20-50 monomers) binds to the origin, causing the double-stranded DNA to separate

3. Replication Bubble

• The two complementary strands separate at the origin, forming a bubble
• Active DNA synthesis occurs at the replication fork

4. RNA Primer

• A short RNA fragment (~5-50 nucleotides) is needed to start synthesis
• Enzyme primase (a specific RNA polymerase) + SSB proteins form the primosome
• The primosome produces RNA primers
• Leading strand needs only one RNA primer
• Lagging strand requires constant synthesis and supply of RNA primers

5. DNA Synthesis - Semidiscontinuous and Bidirectional

• All DNA synthesis proceeds in the 5'→3' direction
• Leading strand (continuous/forward strand): synthesis is continuous
• Lagging strand (discontinuous/retrograde strand): synthesis is discontinuous
• Short pieces of DNA (15-250 nucleotides) are made on the lagging strand
• Synthesis is bidirectional from the point of origin

6. Replication Fork and Key Enzymes

• DNA helicases - bind to both strands at the fork; unwind/separate the helix (like a "zip opener"); require ATP
• SSB proteins - stabilize separated strands and provide template for new DNA

7. DNA Synthesis by DNA Polymerase III

• Catalyzes synthesis in the 5'→3' direction (antiparallel to template)
• Requires all four dNTPs: dATP, dGTP, dCTP, dTTP
• Each incoming deoxyribonucleotide is added to the 3' end of the growing chain
• A molecule of pyrophosphate (PPi) is removed with each nucleotide addition

8. Polarity Problem and Okazaki Pieces

• Leading strand (3'-OH oriented toward fork) can be elongated continuously
• Lagging strand faces a problem - no DNA polymerase can add nucleotides toward the 5' end
• Solution: lagging strand is synthesized as small fragments (Okazaki pieces) in the 5'→3' direction, later joined together
• Okazaki pieces = small, discontinuously synthesized DNA fragments produced on the lagging strand of the parent DNA
• DNA Pol I and DNA ligase join these fragments

9. Supercoils and Topoisomerases

• As the double helix separates, supercoils form at the other side
• Type I DNA topoisomerase - cuts the single DNA strand (nuclease activity) then reseals it (ligase activity); overcomes supercoiling
• Type II DNA topoisomerase (DNA gyrase) - cuts both strands and reseals them; also overcomes supercoils
• Topoisomerases are targeted by drugs: camptothecin (Type I), amsacrine and etoposide (Type II) - used in treatment of cancers

10. Replacement of RNA Primer by DNA Polymerase I

• New DNA strand synthesis continues until it is close to the RNA primer
• DNA Polymerase I removes the RNA primer and catalyzes synthesis (5'→3') of a DNA fragment that replaces it
• DNA ligase then catalyzes the phosphodiester bond linking the fragments (requires energy from ATP to AMP + PPi)

11. Proof-Reading by DNA Polymerase III

• DNA Pol III has a proof-reading activity
• Checks incoming nucleotides; only allows correctly matched (complementary) bases to be added
• Edits and removes wrongly placed nucleotide bases

REPLICATION IN EUKARYOTES

Key Differences from Prokaryotes

Eukaryotic DNA Polymerases

1. DNA polymerase α (alpha) - responsible for synthesis of RNA primer; works on both leading and lagging strands
2. DNA polymerase β (beta) - involved in DNA repair; function comparable to Pol I in prokaryotes
3. DNA polymerase γ (gamma) - replication of mitochondrial DNA
4. DNA polymerase δ (delta) - responsible for replication on the leading and lagging strands; has proof-reading activity
5. DNA polymerase ε (epsilon) - involved in proof-reading of DNA replication

Process on the Leading Strand (Eukaryotes)

• Relatively simple
• Involves DNA polymerase δ + a sliding clamp: PCNA (Proliferating Cell Nuclear Antigen)
• PCNA forms a ring around DNA to which Pol δ binds
• Ring formation requires Replication Factor C (RFC)

Process on the Lagging Strand (Eukaryotes) - More Complex

1. Parental strands separated by helicase
2. Replication Protein A (RPA) (single-stranded DNA binding protein) binds to exposed single-stranded template
3. Primase forms a complex with DNA Pol α → initiates Okazaki fragments; capable of producing a 10-bp RNA primer
4. Activity switches from primase to DNA Pol α, which elongates the primer by adding 20-30 deoxyribonucleotides
5. Thus, a short stretch of DNA attached to RNA is formed, and the complex dissociates from DNA
6. Replication Factor C (RFC) (clamp loader) binds to the elongated primer and catalyzes assembly of PCNA (sliding clamp)
7. DNA Pol δ binds to the PCNA ring and elongates the Okazaki fragment to a final length of ~150-200 bp
8. As elongation approaches the RNA primer of the previous Okazaki fragment, RNA primer removal is carried out by:
   • RNase H and Flap Endonuclease I (FEN1)
9. The gap is filled by continued elongation of the new Okazaki fragment by Pol δ
10. The small remaining nick is sealed by DNA ligase

Chromatin and Histones During Replication

• Eukaryotic DNA is tightly bound to histones (basic proteins) forming nucleosomes, which organize into chromosomes
• During replication, chromosomes are relaxed and nucleosomes loosen
• DNA strands separate; parental histones associate with one of the parental strands
• New histones are also synthesized simultaneously as new DNA is made
• At end of replication: one daughter chromosome has parental histones, the other has newly synthesized histones

Multiple Origins of Replication

• Multiple replication bubbles form in eukaryotic DNA
• Essential for rapid replication of large eukaryotic genomes

INHIBITORS OF DNA REPLICATION

CELL CYCLE AND DNA REPLICATION

• Cell cycle has four distinct phases: Mitotic, G₁, S, and G₂
• When not growing: G₀ (dormant/undividing phase)
• G₁ phase: active protein synthesis
• DNA replication occurs in S phase

Remember: DNA replication differences between bacteria and human cells are exploited in antibacterial therapy - drugs can target pathogen (bacterial) replication enzymes while sparing host (human) cell enzymes.