DNA Metabolism

Bacterial and eukaryotic DNA ligases differ in their energy sources:

Bacterial DNA ligases:

• Use NAD+ as energy source
• Release nicotinamide mononucleotide (NMN) during adenylylation
• Evolved to utilize this cofactor specifically in prokaryotic systems
• E. coli and most bacteria use this mechanism

Eukaryotic DNA ligases:

• Use ATP as energy source
• Release pyrophosphate (PPi) during adenylylation
• Common in viruses and all eukaryotic cells
• More evolutionarily widespread mechanism

This difference represents a fundamental metabolic adaptation and provides a potential target for antibacterial drugs that could inhibit bacterial DNA ligases without affecting human enzymes.

TLS polymerases in prokaryotes: • DNA polymerase IV (dinB gene) and polymerase V (umuC/umuD genes) • Induced during SOS response to severe DNA damage • Lack proofreading exonuclease activity • Low fidelity (error rate ~1 in 1,000 nucleotides) • Primarily function in crisis situations to bypass lethal lesions

TLS polymerases in eukaryotes: • Multiple specialized polymerases (η, ι, κ, Rev1, etc.) • More integrated into normal DNA metabolism • Have specialized roles:

• Pol η primarily bypasses cyclobutane T-T dimers with high accuracy
• Pol ι and κ have roles in base-excision repair
• Many have additional enzymatic activities (e.g., 5'-deoxyribose phosphate lyase) • Often operate on very short DNA stretches (sometimes just 1 nucleotide) • Error rates minimized by context-specific recruitment and limited DNA synthesis • Some function in programmed mutagenesis (e.g., somatic hypermutation in immune cells)

Key difference: Eukaryotic TLS polymerases are more specialized and integrated into normal DNA metabolism rather than just emergency responses.

The RecBCD enzyme:

• Binds to broken DNA ends and moves inward along the double helix
• Has both helicase activity (unwinding DNA) and nuclease activity (degrading DNA)
• Uses ATP hydrolysis to power its movement

When RecBCD encounters a chi sequence (5'-GCTGGTGG-3'):

• Nuclease activity on the strand with the 3' end is greatly reduced
• Degradation of the 5'-terminal strand is increased
• This generates a single-stranded DNA with a 3' end suitable for RecA binding

Chi sequences enhance recombination frequency 5-10 fold within 1,000 bp of the chi site, with diminishing effect as distance increases.

RecA-mediated DNA strand exchange proceeds through five key steps:

1. RecA protein monomers assemble cooperatively on single-stranded DNA, forming a helical nucleoprotein filament
2. This RecA-ssDNA filament incorporates a homologous duplex DNA, forming a three-stranded pairing intermediate
3. ATP-dependent spooling action causes rotation of the DNA molecules, allowing one strand from the duplex to transfer to the original single strand
4. Branch migration proceeds with continued ATP hydrolysis, extending the heteroduplex region while displacing the other strand
5. Complete strand exchange results in a new heteroduplex DNA within the filament and a fully displaced single strand

Note: This process is directional (5'→3' relative to the single strand in the filament) and proceeds at approximately 6 base pairs per second.

Bacteriophage λ integrase (INT) achieves site-specific recombination through:

1. Recognition of specific attachment sites:

   • attP (phage) and attB (bacterial) sites share only 15 bp of homology
   • INT recognizes the unique architecture of these sites rather than extensive sequence identity

2. Mechanism for limited homology sites:

   • INT binds to specific DNA sequences at attachment sites
   • Creates precise double-strand breaks at the crossover region
   • Forms covalent protein-DNA intermediates during strand exchange
   • Rejoins the DNA strands in new configurations with high fidelity

3. Auxiliary protein assistance:

   • IHF (integration host factor) bends the DNA, bringing distant sites into proximity
   • Creates optimal 3D geometry for the recombination reaction

This precision allows bacteriophage λ to insert its entire genome into a specific location in the E. coli chromosome without disrupting essential bacterial genes, demonstrating how highly specialized recombination can occur without extensive homology.

During bacteriophage λ integration, the following new DNA structures are created:

1. New attachment sites:

   • attL: Left junction between prophage and bacterial DNA
   • attR: Right junction between prophage and bacterial DNA
   • These sites represent hybrid sequences containing elements from both original attachment sites

2. Functional significance:

   • The integrated prophage (λ DNA inserted into the bacterial chromosome) replicates passively with the host
   • attL and attR serve as recognition sites for the excision machinery
   • The arrangement provides molecular "memory" of the integration event
   • The prophage state allows bacteriophage genes to be silenced while maintaining the potential for later excision

3. Life cycle importance:

   • Enables the lysogenic pathway where phage DNA is maintained without killing the host
   • Provides environmental persistence strategy through host survival
   • Creates genetic switch that can be triggered by cellular stress (UV damage, etc.) to initiate excision and return to lytic cycle

This elegant system demonstrates how precise DNA rearrangements can create reversible genetic states that benefit both the phage (long-term survival) and potentially the host (immunity from superinfection by similar phages).

Transposases:

• Function: Mobilize transposable elements within genomes
• Mechanism: Cleave at transposon ends and catalyze integration at target sites
• Target specificity: Often low, with some sequence preferences
• Outcomes: Transposon movement or replication, sometimes creating genomic mutations
• Examples: Bacterial IS element transposases, Tn5 transposase

Site-specific recombinases:

• Function: Catalyze recombination between specific DNA sequences
• Mechanism: Make staggered cuts at specific sites and rejoin partners in new configuration
• Target specificity: Very high, recognize specific sequences
• Outcomes: Integration, excision, inversion, or resolution of DNA segments
• Examples: λ integrase, Cre recombinase, Flp recombinase

RAG1/RAG2 proteins:

• Function: Catalyze V(D)J recombination in immune system development
• Mechanism: Create double-strand breaks via hairpin intermediates
• Target specificity: High, recognize recombination signal sequences (RSS)
• Outcomes: Programmed deletion of DNA between coding segments
• Evolutionary origin: Share biochemical features with transposases

Key similarities and differences:

• All create DNA breaks and rejoin DNA strands
• Transposases and RAGs create hairpin intermediates; recombinases typically don't
• RAGs and site-specific recombinases target specific sequences; transposases are less specific
• Transposases and RAGs can catalyze transposition in vitro; site-specific recombinases cannot
• RAGs function exclusively in vertebrate immune system; others are widely distributed

Mechanism of Target Site Duplication:

1. Transposase creates staggered (offset) cuts in the target DNA, producing sticky ends
2. The transposon is inserted between these staggered cuts
3. DNA polymerase fills in the single-stranded gaps
4. DNA ligase seals the remaining nicks in the phosphodiester backbone
5. The result is a duplication of the short target site sequence flanking both sides of the inserted transposon

Required Enzymes:

• Transposase (for cutting and strand transfer)
• DNA polymerase (for gap filling)
• DNA ligase (for sealing nicks)

Biological Significance:

• Creates characteristic "footprints" that allow identification of transposition events
• May provide buffer zones between transposon and adjacent DNA sequences
• Serves as recognition sites for future transposition events in some systems
• Preserves genomic content during the insertion process

DNA polymerase III overcomes the directional challenge of antiparallel DNA strands through a sophisticated asymmetric architecture:

Structure and Organization

• Dimeric architecture: Contains two identical core polymerases (α, ε, θ) connected by τ subunits
• Clamp loader complex: γ complex loads β clamps at primer sites
• DnaB helicase: Travels along lagging strand template (5'→3') unwinding DNA ahead of replication

Coordination Mechanism

1. DNA looping arrangement:

   • Leading strand: Synthesized continuously in the same direction as fork movement
   • Lagging strand: Template forms a loop allowing synthesis in physical direction opposite to fork movement
   • Both polymerases physically move in the same direction despite synthesizing antiparallel strands

2. Lagging strand dynamics:

   • Discontinuous synthesis of Okazaki fragments
   • Polymerase recycling: Core releases from completed fragments and reinitiates at new primers
   • Coordinated loading of new β clamps at RNA primers

Functional Advantages

• Maintains equal synthesis rates on both strands
• Preserves processivity despite discontinuous lagging strand synthesis
• Enables a single replisome to efficiently handle both strands simultaneously
• Solves the inherent directional constraints of 5'→3' synthesis on antiparallel templates

Note: This tethered arrangement explains how replication can proceed at equal rates on both strands despite their opposite polarities—a key solution to what would otherwise be a fundamental biochemical paradox.

DNA polymerase requires multiple essential components that collectively ensure accurate DNA replication:

1. Template DNA strand:

   • Must be read in the 3'→5' direction
   • Provides sequence information via complementary base-pairing
   • Represents the first known example of template-guided biosynthesis
   • Enables semiconservative replication pattern

2. Primer with free 3'-OH group:

   • DNA polymerases cannot initiate synthesis de novo
   • 3'-OH serves as the nucleophile for the addition reaction
   • In vivo, RNA primers are synthesized by primase
   • Explains why lagging strand requires multiple primers
   • Highlights the interdependence of RNA and DNA synthesis

3. Incoming dNTPs:

   • Supply nucleotide building blocks in triphosphate form
   • Provide energy for polymerization through phosphodiester bond formation
   • Selection governed by Watson-Crick base pairing rules

4. Two Mg²⁺ ions:

   • Coordinate with conserved aspartate residues in the active site
   • Essential for catalysis and proper substrate orientation

5. Appropriate active site geometry:

   • Accommodates only properly paired bases
   • Contributes significantly to replication fidelity (error rate ~10⁻⁴ to 10⁻⁵)

These requirements collectively enable both catalytic efficiency and high accuracy, which are essential for maintaining genetic information integrity across generations and explain why DNA replication requires a coordinated system of multiple enzymes.