DNA Replication

Biochemistry II: DNA Replication Notes

The Central Dogma of Molecular Biology

• The central dogma describes the flow of genetic information in cells.
• DNA functions:
  • Directs its own replication.
  • Directs transcription of RNA (gene expression).

DNA Replication: Identical Copies

• DNA replication results in identical copies of chromosome pairs in each daughter cell.

Conservative vs. Semiconservative Replication

• Two possible models for DNA replication:
  • Conservative: The original double-stranded DNA molecule remains intact, and a completely new DNA molecule is synthesized.
  • Semiconservative: The double-stranded DNA molecule splits into two, and each strand acts as a template for a new complementary strand. This results in two DNA molecules, each containing one original and one new strand.

1958 Messelson and Stahl Experiment

• Background:
  • Grew E. coli in a medium containing $^{15}NH_4Cl$ to produce $^{15}N$-DNA (heavy).
  • Grew E. coli in a medium containing $^{14}NH_4Cl$ to produce $^{14}N$-DNA (light).
  • Monitored DNA density using equilibrium gradient centrifugation.

Experimental Procedure

• Parental E. coli grown in $^{15}NH_4Cl$.
• One generation after switching to $^{14}NH_4Cl$.
• Two generations after switching to $^{14}NH_4Cl$.

Conclusion of the Experiment

• The experiment provided evidence that DNA replication is semiconservative.

DNA Replication by DNA Polymerases

• DNA is replicated by DNA polymerases.
• DNA polymerases:
  • Work only in the 5' → 3' direction.
  • Use single-stranded DNA (ssDNA) as templates to synthesize a complementary DNA strand.
  • Require incoming nucleotides to form Watson-Crick base pairs with the template.
  • Link nucleotides via phosphodiester bonds.
  • Require an RNA primer to provide a free 3'-OH group. The primer is synthesized by RNA polymerase or primase (dnaG).
  • Cannot initiate DNA synthesis from free nucleotides.

SEmidiscontinuous Replication

• Replication is semidiscontinuous
• The two parent strands are replicated in different ways:
  • Leading strand: Replicated continuously in the 5' → 3' direction.
  • Lagging strand: Replicated discontinuously in the 5' → 3' direction as Okazaki fragments.
    • Okazaki fragments:
      • The 5' end consists of RNA primers (1-70 nucleotides) synthesized by primase.
      • The Okazaki fragment is extended in the 5' → 3' direction.
      • A new primer forms near the replication fork, and a new Okazaki fragment is extended 5' → 3'.
      • Primers are excised by Pol I (5' → 3' exonuclease activity).
      • The gap is filled in by Pol I, and the nick is sealed by ligase.

E. Coli DNA Polymerases

• Three types: Pol I, Pol II, Pol III.
• All three have 5' → 3' polymerase activity.
• Pol III: Primary enzyme for DNA replication (1000 nucleotides/sec).
• All have 3' → 5' exonuclease activity; excises an incorrect nucleotide at the end of a growing DNA chain (proofreading).
• Only Pol I has 5' → 3' exonuclease activity but has low polymerase activity (10 nucleotides/sec); removes RNA primers and is used in DNA repair.

Events of Replication of E. coli

• Initiation is precise: once per cell division.
• Has only one origin of replication (eukaryotes have multiple origins/chromosome).
• Inactive origin → active origin by the binding of initiation proteins that bend open the double helix (helicase that uses ATP).

The Replisome

• The synthesis of both strands occurs in a replisome (multi-protein complex).
• Components:
  1. Gyrase (Topo II): Unwinds the DNA.
  2. Rep protein (leading strand) & Helicase II (lagging strand): Separate base pairs.
  3. Single-stranded binding protein (SSB): Keeps the strands separated.
  4. Primase and RNA Pol: Makes primers.
  5. DNA Pol III: Synthesizes DNA.
  6. DNA Pol I: Removes primers.
  7. DNA ligase: Links Okazaki fragments.

Topoisomerases

• Enzymes that alter the linking number of DNA and unwind it.
• Linking number: The number of times one strand winds around the other in a right-handed direction = changes in supercoiling.
• Type I Topo: Cleaves one strand; no energy required.
• Type II Topo: Cleaves both strands; requires energy.

Eukaryotic DNA Polymerases

• DNA is replicated in the S phase of the cell cycle.
• Four types:
  • DNA pol α: Role in replication of chromosomal DNA in the nucleus; multi-subunit with primase activity; lacks exonuclease activity (no proofreading); lagging strand replicase.
  • DNA Pol δ: Leading strand replicase; lacks primase activity; has 3' → 5' exonuclease activity.
  • DNA Pol β: Involved in DNA repair.
  • DNA Pol γ: Role in Mitochondrial DNA replication.

DNA Repair

• DNA damage occurs spontaneously and can be induced by environmental factors such as chemicals and UV radiation.
• Types of repair:
  • Direct Repair: Reversal of damage using enzymes.
    • Example: Pyrimidine dimers ($T=T$) are repaired by photolyase ($T=T osto T + T$).

Nucleotide Excision Repair

• Steps:
  1. An endonuclease cleaves the DNA strand on both sides of the lesion (E.coli- UVrABC endo).
  2. Excision of about 12 nucleotides by 5'→ 3' exo of Pol I.
  3. Gap is filled by Pol I.
  4. DNA ligase seals the nick.
• Xeroderma Pigmentosum: A rare inherited disease with defects in Nucleotide Excision Repair.
  • Causes patients to have a 1000-fold increased risk for skin cancer.

Telomeres

• G-rich DNA repeats + proteins at the end of chromosomes.
• Protect against degradation and recombination.
• Not fully replicated by DNA polymerase → they shorten with each round of cell division.

Telomere Hypothesis

• Telomeres Regulate Cellular Life Span. Aging control.
• Telomerase: Enzyme that maintains telomere length.
  • Ribonucleoprotein:
    • RT (reverse transcriptase)
    • RNA template
    • Proteins
• Tumor cells have an increase of telomerase activity.
• Stem cells have an increase of telomerase activity.

Nucleic Acid Structure

• Deoxyribose:

• Ribose:

DNA and RNA Components

• DNA:
  • Nitrogenous bases: Adenine (A), Guanine (G), Thymine (T), Cytosine (C)
  • Sugar: Deoxyribose
• RNA:
  • Nitrogenous bases: Adenine (A), Guanine (G), Uracil (U), Cytosine (C)
  • Sugar: Ribose

Fish Chromosome Packing Ratio

• A fish has an average chromosomal length of 2.5 cm. During metaphase, each chromosome is 25 microns.
• What is the packing ratio?
  $Packing Ratio = \frac{2.5 cm}{25 \mu m} = \frac{2.5 \times 10^4 \mu m}{25 \mu m} = 1,000$