DNA Replication Part 2: Elongation, Enzymes, Energetics, and Mutation Mechanisms

Principles of DNA Chain Elongation

• Basics of DNA Synthesis: The process of elongation involves adding deoxyribonucleotides one at a time to the free $3'$ end of a growing DNA strand. The direction of synthesis is always written as $5'$ to $3'$
• The Role of DNA Polymerases: These enzymes are responsible for cataloging and adding the correct bases. In prokaryotes, DNA Polymerase III ($DP III$) performs the vast majority of the work.

Characterization of DNA Polymerase I (DPI)

• Discovery: DNA Polymerase I was the first to be discovered because it exists in much higher concentrations within the cell than subsequent polymerases.
• Efficiency and Speed:
  • $DPI$ is relatively inefficient, adding only $20$ to $50$ new bases before physically falling off the parent strand.
  • Its overall activity rate is approximately $600\text{ bases/min}$.
  • At this rate, replicating a single E. coli chromosome would take approximately $25$ hours, which is far too slow for biological requirements.
• Enzymatic Activities: $DPI$ possesses three distinct enzymatic activities:
  1. $5' \rightarrow 3'$ DNA Polymerase: The ability to read the template DNA and write the new DNA strand.
  2. $3' \rightarrow 5'$ Exonuclease: Often referred to as the "Backspace" function. It allows the enzyme to step backward, remove an incorrectly paired base, and then resume synthesis.
  3. $5' \rightarrow 3'$ Exonuclease: Often referred to as the "Typeover" function. It is used to read over the RNA primer, remove it, and replace it with the appropriate DNA sequence.
• Structural Note: The "Klenow Fragment" refers to the portion of $DPI$ that retains the polymerase and $3' \rightarrow 5'$ exonuclease activity but lacks the $5' \rightarrow 3'$ exonuclease activity. This makes it structurally similar to $DP III$.
• Functional Focus: $DPI$ is primarily utilized on the lagging strand to handle the removal and replacement of primers for Okazaki fragments.

DNA Polymerase II (DPII) and DNA Polymerase III (DPIII)

• DNA Polymerase II (DPII):
  • Principally involved in DNA repair mechanisms.
  • Lacks the $5' \rightarrow 3'$ exonuclease activity.
  • Its primary function is to perform proofreading between replication cycles.
• DNA Polymerase III (DPIII):
  • The last to be discovered due to its low concentration in the cell.
  • High Velocity: It adds approximately $1,000\text{ bases/second}$.
  • Efficiency Comparison: A single molecule of $DP III$ could replicate the $4.5 \times 10^6$ bases in an E. coli strand in about $75$ minutes (as opposed to the $5$ days it would theoretically take $DPI$ if it worked alone).
  • Function: It connects to the primer and lays down many bases very quickly. It is the primary enzyme for the leading strand.
  • Movement: As it moves, it "bumps off" Single-Strand Binding proteins ($SSBs$). The newly added base then stabilizes the parent strand base.
  • Exonuclease Activity: Like the Klenow fragment, it has $3' \rightarrow 5'$ exonuclease (proofreading) but lacks $5' \rightarrow 3'$ exonuclease activity because it is not responsible for primer removal.
• Note on Primase: Unlike DNA polymerases, Primase has no exonuclease activity. Mistakes in the RNA primer do not matter because $DPI$ eventually removes and replaces the entire sequence.

Energetics and Final Ligation

• Energy Consumption: The formation of every $3'-5'$ phosphodiester bond (each base addition) requires high energy.
  • The process uses approximately $2\,ATP$ equivalents per base.
  • The reaction is coupled: $ATP + DNA \rightarrow DNA + \text{Pyrophosphate}$.
  • Both high-energy bonds in $ATP$ are hydrolyzed. An inorganic pyrophosphatase enzyme breaks down the resulting pyrophosphate ($PP_i$) to ensure the preceding reaction is energetically favorable and proceeds forward.
• DNA Ligase: Once $DPI$ replaces the RNA primers with DNA, the newly synthesized strand is made entirely of DNA, but the Okazaki fragments are not yet covalently bonded to one another.
  • DNA Ligase is the enzyme that ties these ends together.
  • This is an $ATP$-dependent process that forms the final phosphodiester bond to create a continuous strand.

Eukaryotic Replication and Organellar DNA

• Origins of Replication ($ori$): Unlike prokaryotes with a single origin, eukaryotes have many origins of replication.
• Replisome Spacing: Replisomes are spaced approximately $10,000$ to $30,000$ bases apart and move toward each other during synthesis.
• Histones: These positively charged proteins used for DNA compaction must be synthesized during replication. In the resulting daughter cells, one set of DNA retains the old histones while the other set receives the newly synthesized histones.
• Mitochondrial DNA (mtDNA):
  • Mitochondria were originally endosymbiotic prokaryotic cells.
  • They contain a single circular chromosome and a genetic code that differs from nuclear DNA.
  • Maternal Inheritance: All mitochondrial DNA is inherited from the mother (via the egg).
  • Lineage Tracking: This maternal inheritance allows for tracking ancestral lines.
  • Example: The remains of Czar Nicholas and Alexandra of Russia were identified using mitochondrial DNA comparisons between bone samples and living maternal descendants.

Types and Causes of Mutations

• Mutation Rates: In $T4\text{ Phage}$, mutations occur at a rate of approximately $1$ in $10^7$ ($10$ million) bases. Rates in eukaryotes are significantly lower but still occur.
• Substitution Mutations: Replacing one base pair with another across generations.
  • Transition: Substituting one purine for the other ($A \rightleftharpoons G$) or one pyrimidine for the other ($C \rightleftharpoons T$).
  • Transversion: Substituting a purine for a pyrimidine or vice versa.
• Deletions: The loss of a base pair in future generations, often causing a frameshift (e.g., $AAAGG \rightarrow TTTCC$ losing a base and shifting the sequence).
• Insertions: The appearance of a new base pair in future generations, also causing a frameshift.
• Chemical Mutagens:
  • Analogs: Molecules like $5\text{-bromouracil}$ are thymine analogs. They substitute for thymine but can cause an $A-T$ pair to transform into a $C-G$ pair (a transversion).
  • Alkylating Agents: Examples include mustard gas (used in WWI). These alter base structures and typically result in transversions.
  • Interference (Intercalating Agents): Planar molecules like acridine dyes or Quinine (an antimalarial) insert themselves between bases. This "intercalation" typically produces insertions and deletions.
• Ultraviolet (UV) Light: The energy from UV light (e.g., sunburn) is absorbed by DNA and causes:
  1. Spontaneous deamination of Cytosine ($C$) to Uracil ($U$).
  2. Thymine residues can replace the Uracils and dimerize (forming $T-T$ dimers), which present significant problems for the next round of replication.
• Impact of Mutations: Mutations may have no effect, or they may block replication, or change the DNA code in a way that alters transcription and translation.