DNA Metabolism

DNA synthesis must be semi-discontinuous because:

1. DNA polymerase can only synthesize DNA in the 5'→3' direction
2. The two DNA strands in the double helix are antiparallel (run in opposite directions)
3. The replication fork moves in one direction while both strands must be replicated simultaneously

This constraint is resolved through:

• The leading strand: Synthesized continuously in the 5'→3' direction, matching the direction of fork movement
• The lagging strand: Synthesized discontinuously as Okazaki fragments in 5'→3' direction, opposite to fork movement
• Okazaki fragments are later joined by DNA ligase

In bacteria, Okazaki fragments are ~1,000-2,000 nucleotides long; in eukaryotes, they're 150-200 nucleotides long.

Tautomeric shifts in nucleotide bases lead to replication errors through:

1. Rare tautomeric forms (e.g., cytosine C* instead of normal C) temporarily alter hydrogen bonding properties
2. These altered forms can pair with incorrect bases (e.g., C* pairs with A instead of G)
3. After incorporation, the base reverts to its common tautomeric form, creating a mismatch

Detection occurs because:

• The resulting mismatched base pair creates geometric distortion in the DNA helix
• This distortion inhibits translocation of DNA polymerase to the next position
• The kinetic pause allows time for the proofreading mechanism to detect and correct the error
• The incorrect nucleotide becomes accessible to the 3'→5' exonuclease active site

This detection system ensures that even errors caused by temporary chemical states of nucleotides can be identified and fixed.

The coordination involves:

1. DNA looping: Lagging strand template is looped so both leading and lagging strand synthesis occur in same direction
2. Core cycling: Two core polymerase units in DNA polymerase III work together:
   • One continuously synthesizes leading strand
   • The other cycles between multiple Okazaki fragments on lagging strand
3. Primosome activity: DnaB helicase and DnaG primase work as functional unit
   • DnaB moves 5'→3' on lagging strand template, unwinding DNA
   • Primase occasionally associates with helicase to synthesize RNA primers
4. Clamp management:
   • New β sliding clamps positioned at each primer by clamp-loading complex
   • When Okazaki fragment completes, core polymerase detaches from old clamp
   • Core then associates with new clamp at next primer site
5. Physical coupling: Entire replisome remains physically associated, ensuring coordination

DNA polymerase I functions:

• 5'→3' polymerase activity (adds nucleotides to 3' end)
• 3'→5' exonuclease activity (proofreading)
• 5'→3' exonuclease activity (removes RNA primers, critical for Okazaki fragment maturation)

Nick translation process:

1. Begins at a nick (broken phosphodiester bond with free 3'-OH and 5'-phosphate)
2. DNA polymerase I uses 5'→3' exonuclease activity to remove nucleotides ahead
3. Simultaneously, polymerase activity adds nucleotides to the 3'-OH end
4. The nick "moves" or "translates" along the DNA as this occurs
5. This process continues until polymerase I dissociates, leaving a nick
6. Final nick is sealed by DNA ligase

This mechanism is vital for both DNA repair and RNA primer removal during replication.

Both ligases seal nicks in DNA through a three-step process:

Step 1: Enzyme adenylylation

• Bacterial ligase: Uses NAD⁺ as cofactor, releasing nicotinamide mononucleotide (NMN)
• Eukaryotic/viral ligase: Uses ATP as cofactor, releasing pyrophosphate (PPᵢ)

Step 2: Transfer of AMP to 5' phosphate

• Both: AMP from enzyme-adenylyl intermediate transfers to 5' phosphate at nick
• Forms activated 5'-adenylyl-phosphate intermediate

Step 3: Nick sealing

• Both: 3'-OH attacks the activated 5' phosphate
• Displaces AMP and forms phosphodiester bond
• Releases AMP as byproduct

Key difference: Energy source (NAD⁺ vs ATP), reflecting the evolutionary divergence while maintaining the essential chemistry of DNA ligation.

Bacterial DNA polymerase III has a complex architecture with multiple functional components:

• Two core domains (α, ε, θ subunits) that perform the primary polymerase and proofreading functions • A five-subunit γ complex (γ₂δδ'χψ) known as the clamp-loading complex that loads the β clamps onto DNA • Two β clamps (each a dimer of β subunits) that encircle the DNA and slide along it • The τ subunits connect the core polymerase to the clamp-loading complex • DnaB helicase interacts with the τ subunit

The τ and γ subunits are encoded by the same gene, with τ being a longer version containing an additional domain that interacts with the core polymerase.

Example: This architecture enables coordinated leading and lagging strand synthesis, with one core working continuously and the other processing Okazaki fragments.

Prokaryotic (E. coli) Initiation:

• Origin: Single oriC (245 bp) with three 13-bp repeats and four 9-bp repeats
• Origin recognition: DnaA protein binds to 9-bp repeats
• Helicase: DnaB (hexameric ring structure)
• Helicase loader: DnaC protein
• Initiation sequence: DnaA-ATP complex → DNA unwinding → DnaC loads DnaB

Eukaryotic Initiation:

• Origins: Multiple replicators/ARS (~400 in yeast, thousands in humans)
• Origin recognition: Origin Recognition Complex (ORC, multi-subunit)
• Helicase: MCM complex (hexamer of MCM2-7 proteins)
• Helicase loaders: CDC6 and CDT1 proteins
• Initiation sequence: ORC binding → CDC6/CDT1 recruitment → MCM loading

Key differences:

• Single vs. multiple origins
• Simpler vs. more complex protein complexes
• Direct cell cycle control in eukaryotes through CDK regulation
• Slower fork movement in eukaryotes (~50 nucleotides/s vs. ~1,000 in E. coli)

The E. coli replication origin (oriC) contains two essential repeated sequence elements:

1. Three tandem 13 bp sequences

   • Consensus sequence: GATCTNTTNTTTT
   • Rich in A-T pairs, making them easier to unwind
   • All oriented in the same direction

2. Four 9 bp sequences

   • Consensus sequence: TTATCCACA
   • Serve as binding sites for DnaA protein
   • Arranged in various orientations throughout the 245 bp oriC region

These sequences work together during replication initiation: DnaA proteins bind to the 9 bp sequences, then facilitate unwinding of the DNA at the A-T rich 13 bp sequences to begin the replication process.

AlkB repair mechanism: • AlkB is an α-ketoglutarate-Fe²⁺-dependent dioxygenase • Repairs alkylation damage that occurs mainly in single-stranded DNA regions • Targets 1-methyladenine and 3-methylcytosine lesions

Reaction components: • Substrate: methylated base (1-methyladenine or 3-methylcytosine) • Cofactors: Fe²⁺, O₂, and α-ketoglutarate

Mechanism:

1. Fe²⁺ coordinates with α-ketoglutarate and the methyl group of the damaged base
2. Oxygen binds and oxidative decarboxylation of α-ketoglutarate occurs
3. This generates a high-valent iron-oxo intermediate
4. The iron-oxo species hydroxylates the methyl group
5. The unstable hydroxymethyl group spontaneously decomposes
6. Releases formaldehyde (CH₂O) and restores the normal base
7. α-Ketoglutarate is converted to succinate in the process

This direct reversal mechanism preserves the original DNA sequence.

Mechanism:

1. Recombinase binds to specific recombination sites on two DNAs
2. One DNA strand in each site is cleaved by nucleophilic attack from Tyr residue
3. Covalent 3'-phosphotyrosine linkage forms, preserving energy
4. Cleaved strands exchange partners, forming a Holliday intermediate
5. Process repeats with the second strand at each site
6. Original recombination site sequence is regenerated after crossover

Outcomes based on site orientation:

• Sites in opposite orientation on same DNA → inversion of intervening DNA
• Sites in same orientation on same DNA → deletion of intervening DNA
• Sites on different DNA molecules → integration/insertion

Example: λ phage integrase mediates integration (insertion) into bacterial chromosome using attP and attB sites, with different protein requirements for integration versus excision.