Genes and Chromosomes

Strained DNA:

• Occurs immediately after underwinding (e.g., removal of helical turns)
• Energetically unfavorable with high thermodynamic strain
• Fewer helical turns per base pair (e.g., 12.0 bp/turn vs. normal 10.5 bp/turn)
• Transient state that quickly converts to more stable forms
• Biologically unstable and rarely persists in cells

Supercoiled DNA:

• Primary mechanism for accommodating underwinding strain
• Energetically more favorable than strained linear form
• Forms when DNA axis coils upon itself, creating superhelical turns
• Requires less energy than breaking hydrogen bonds
• Biologically essential for DNA packaging and regulation
• Maintained by topoisomerases that regulate linking number

Strand-separated DNA:

• Alternative mechanism for relieving underwinding strain
• Energetically more costly than supercoiling (requires breaking hydrogen bonds)
• Creates localized regions where DNA strands are unpaired
• Biologically crucial for processes requiring access to DNA information
• Essential for replication initiation, transcription, and recombination
• Often limited to specific sequences where strand separation is functionally important

Relationship: Underwinding introduces strain → primarily accommodated by supercoiling → facilitates localized strand separation where biologically necessary → all states regulated by topoisomerases to maintain proper DNA topology.

• Relaxed plasmid DNA appears as a loose, open circular structure with minimal folding
• Supercoiled plasmid DNA shows distinct loops, twists, and compact folding patterns
• As supercoiling increases, the DNA structure becomes progressively more compact and organized into defined interwound segments
• The overall contour length appears shorter in supercoiled DNA due to the compaction from writhe
• Supercoiled DNA occupies less physical space than its relaxed counterpart of identical sequence length

Example: In bacterial cells, plasmids typically exist in a negatively supercoiled state, which is maintained by topoisomerases and influences numerous cellular processes including transcription and replication.

The 30 nm fiber is:

• The second level of chromatin organization, beyond the nucleosome "beads-on-a-string" arrangement
• A helical structure formed by the coiling of nucleosomes with the help of histone H1
• Approximately 30 nanometers in diameter (hence the name)
• Provides approximately 100-fold compaction of DNA compared to linear DNA
• Organized with multiple nucleosomes positioned into a solenoid-like helical structure

Note: The 30 nm fiber is less prevalent in actively transcribed regions of chromatin, which typically contain little or no histone H1.

Specific linking difference (σ):

• Defined as: σ = ΔLk/Lk₀ (the change in linking number divided by relaxed linking number)
• Also called superhelical density
• Measures the degree of supercoiling per helical turn of DNA
• A quantitative measure of underwinding or overwinding

Significance:

• Negative σ values indicate underwound DNA (most common in nature)
• Prokaryotic DNA typically has σ = -0.06 (6% underwound)
• Eukaryotic DNA in nucleosomes has effective σ ≈ -0.1
• σ affects DNA compaction and energy stored in DNA structure
• Influences binding of regulatory proteins to DNA
• Affects processes like transcription, replication, and recombination
• Can determine success of processes like bacteriophage integration (example: λ phage requires specific σ range for integration)

This parameter provides a universal way to compare supercoiling across different DNA molecules of varying sizes.

Structure and composition:

• Second level of chromatin organization beyond nucleosomes
• ~30 nm in diameter
• Composed of nucleosomes organized into a solenoid
• Each nucleosome contains histone octamer (2× H2A, H2B, H3, H4) + DNA
• Requires histone H1 bound to linker DNA between nucleosomes
• Achieves ~100-fold compaction of DNA

Regulation:

• Formation depends on transcriptional activity of DNA region
• Transcriptionally active regions have little/no histone H1 and less 30 nm fiber structure
• Can be disrupted by histone modifications (especially acetylation)
• Chromatin remodeling complexes can alter the structure

Functional significance:

• Provides intermediate level of DNA compaction
• Creates domains of active versus inactive chromatin
• Serves as foundation for higher-order chromatin structures
• Helps regulate DNA accessibility for transcription
• Contributes to chromosome territory formation in nucleus
• May help organize functional gene clusters

The 30 nm fiber is not uniform throughout the genome but varies with transcriptional activity and cell cycle stage.

• Nucleosomes are the fundamental organizational units of chromatin in eukaryotic cells
• Each nucleosome consists of:
  • A histone core (an octamer of 8 histone proteins: 2 each of H2A, H2B, H3, and H4)
  • ~146 bp of DNA wrapped around the histone core in a left-handed solenoidal supercoil
  • Linker DNA connecting adjacent nucleosomes (varies from 20-80 bp)
• Nucleosomes create a "beads-on-a-string" appearance when viewed with electron microscopy
• This arrangement compacts DNA approximately 7-fold compared to linear DNA
• Regular spacing of nucleosomes forms the first level of DNA packaging in eukaryotes
• Histone H1 binds to the linker DNA to help stabilize higher-order chromatin structures

• DNA wraps around the histone octamer in a left-handed solenoidal supercoil
• This wrapping removes approximately one helical turn from the DNA
• When nucleosome binding occurs on closed-circular DNA:
  • A negative supercoil forms where DNA wraps around the histone core
  • A compensatory positive supercoil forms in the unbound region
  • Topoisomerases can relax the positive supercoil
  • This results in a net negative linking number change (ΔLk = -1)
• The tight wrapping requires compression of the minor groove of the DNA helix
• A⋅T base pairs are often found at contact points as they facilitate minor groove compression

The V-shaped structure forms through:

1. Intramolecular folding: Each SMC polypeptide folds at its hinge domain, bringing N and C terminals together to form a complete ATPase domain
2. Dimerization: Two folded SMC monomers connect at their hinge domains to create the V-shape
3. Flexibility: The coiled-coil regions create dynamic arms that can change conformations

Functional significance:

• Creates a ring-like structure capable of encircling DNA strands
• Enables ATP-dependent conformational changes critical for DNA interaction
• Provides mechanical leverage for manipulating chromosome structure
• Allows spatial separation between DNA-binding sites and ATP hydrolysis regions
• Facilitates diverse functions from sister chromatid cohesion (cohesins) to chromosome condensation (condensins)

This architecture is evolutionarily conserved from bacteria to humans, underscoring its fundamental importance in chromosome dynamics.

1. Autonomously Replicating Sequence (ARS)

   • Functions as an origin of replication
   • Allows the YAC to replicate during S phase
   • Contains DNA unwinding elements and protein binding sites

2. Centromere (CEN)

   • Ensures proper segregation during mitosis
   • Attaches the YAC to spindle fibers
   • Required for equal distribution to daughter cells

3. Telomeres (TEL)

   • Protect chromosome ends from degradation and fusion
   • Prevent the YAC from being recognized as damaged DNA
   • Allow complete replication of chromosome ends

Without any of these three elements, YACs would fail to be maintained stably through multiple cell divisions.

• DNA underwinding (negative supercoiling):

  • Creates torsional strain in the double helix
  • Often results from transcription (as RNA polymerase moves forward)
  • Predominantly occurs in transcriptionally active regions

• Z-DNA formation:

  • Relieves negative supercoiling stress
  • Requires specific sequences (alternating purines/pyrimidines)
  • Left-handed helix with zigzag backbone structure

• Transcriptional connection:

  • Z-DNA often forms near transcription start sites
  • Acts as a topological buffer for supercoiling from transcription
  • May serve as recognition sites for transcription factors
  • Can modulate chromatin structure to expose regulatory elements
  • Found more frequently in actively transcribed regions than silent ones

This relationship illustrates how DNA topology, structure, and function are inextricably linked.