DNA Replication Notes

Polymerization Reactions

• The essential reaction catalyzed by DNA Pol I and III is a polymerization:
  (DNA)n + dNTP \rightarrow (DNA){n+1} + PPi
  (DNA)_{n+1} + 2 Pi
• The 3'-OH of the polymer attacks the innermost (α) phosphate of the incoming NTP, forming a phosphodiester bond.
• Requirements for polymerization:
  • Nucleotides: dATP, dGTP, dCTP, and TTP serve as activated precursors.
  • Mg^{2+} counters the abundant negative charge.
  • DNA template: Complementarity of the incoming base is required for efficient addition.
  • Primer: A strand containing a free 3'-OH group for attack. RNA acts as a primer in vivo.

Specificity and Fidelity in Chain Extension

• Interactions with template DNA promote specificity through:
  • Base pairing between template and incoming NTP, where hydrogen bonds stabilize interactions, and incorrect pairs are relatively unstable.
  • Overall shape complementarity, which can drive the incorporation of bases without H-bonding groups via induced fit, forming a tight pocket with the proper shape.
• Exonuclease activity increases polymerase fidelity by proofreading the newest addition, increasing fidelity by ~1000X.
  • It relies on 3’-to-5’ exonuclease activity.
  • If an incorrect base is added:
    • The polymerase stalls due to structural distortion.
    • Incorrect H-bonding encourages movement.
    • The base is removed upon entering the exonuclease active site.
  • It also removes correct bases (~5%).
  • 3’-to-5’ exonuclease activity is found in DNA Pol I and III.

Helicases and Topoisomerases

• DNA replication requires the separation of strands.
  • DNA helicases are responsible for strand separation.
    • The enzyme is typically a hexameric ring.
    • Repeated ATP hydrolysis pulls one strand through the center.
    • The protein acts as a “wedge” to drive strands apart.
• DNA unwinding in replication introduces topological strain.
  • Underwinding in strand-separation generates compensating overwinding elsewhere.
  • Topoisomerases are enzymes that can relieve the strain of over- or underwound DNA by cleaving DNA and adding/subtracting DNA turns.
• Types of topoisomerases:
  • Topoisomerase 1 (E. coli):
    • Type IA topoisomerase
    • Relaxes DNA by passing an intact strand through a cleaved strand (single-strand break).
    • Reduces DNA underwinding
    • Does not require ATP hydrolysis
  • Gyrase (E. coli):
    • Type II topoisomerase
    • Relaxes DNA by introducing a double-stranded break.
    • Reduces DNA overwinding/introduces underwinding.
    • Requires ATP hydrolysis.
• Single-strand binding proteins (SSBs) prevent base-pairing by binding to strand-separated DNA, preventing the formation of intramolecular hydrogen bonds.

Replication Initiation

• Replication of the bacterial chromosome begins at a unique site (oriC).
  • It contains four binding sites for the protein DnaA.
  • It also contains an array of AT-rich sequences.
• Process:
  • DnaA binds oriC sites and oligomerizes.
  • Local DNA is wrapped around the DnaA complex.
  • DnaB (a helicase) is recruited to the complex.
  • SSB binds strands exposed by DnaB,
    • Preventing the re-formation of the double helix; at this point, a prepriming complex has been formed.
  • Primase synthesizes RNA primer.

Coordination of Strands

• The replication fork is the point at which active replication is happening.
  • It is the junction of strand-separated and still-to-be-separated DNA.
• Both new strands are synthesized concurrently.
  • Leading strand: Synthesis on one strand (leading strand) is in the same direction as fork movement.
  • Lagging strand: Synthesis on the other strand (lagging strand) is opposite to the direction of fork movement.
  • Difficulty arises due to the antiparallel arrangement of strands.
• To achieve net growth in the 3’-to-5’ direction (despite the rule that synthesis is 5’ to 3’), lagging strand DNA is produced in fragments.
• Action on the lagging strand:
  • Primase initiates synthesis at multiple positions, replacing RNA with DNA (not possible for DNA Pol III, which only has 3’-to-5’ activity).
  • DNA polymerase III extends primers to ~1000 nucleotides (Okazaki fragments).
  • DNA polymerase I uses 5’-to-3’ exonuclease activity to remove RNA primers (~10 nucleotides).
  • DNA ligase forms phosphodiester bonds between Okazaki fragments.
• Action on the lagging strand costs two high-energy bonds (ATP \rightarrow AMP)
• Machinery at the replication fork:
  • Helicases: unwinding DNA
  • SSBs: preventing intra-strand base pairing
  • Primase: making RNA primers
  • Polymerases: extending DNA (5’ to 3’)
    • On the leading strand (continuous): DNA polymerase III
    • On the lagging strand (discontinuous): DNA polymerase I and III
  • Nucleases: DNA Pol I (RNA removal, editing); DNA Pol III (editing)
  • Ligase: joining fragments on the lagging strand
  • Clamp loader (not pictured)

DNA Polymerase III

• DNA polymerase III is the major polymerase in DNA synthesis.
• Interactions between proteins support effective replication:
  • Two copies of the core (polymerase) subunit,
    • Each includes a sliding clamp to increase processivity,
    • Each includes an exonuclease subunit (proofreading).
  • Clamp loader
• It can catalyze the addition of 1000 nucleotides / second.
• The core polymerase is assimilated into a multi-subunit holoenzyme and is part of the central structure between core enzymes, interacting with SSB and helicase.
• Holoenzyme assembly keeps strand synthesis coordinated:
  • Two copies of the core (polymerase) subunit.
  • The lagging strand is looped out, growing as the fork proceeds.
  • After DNA Pol III adds ~1000 nucleotides, the template is released.
  • Primase initiates a new fragment, which reattached Pol III extends.

Eukaryotic DNA Replication

• The mechanism of replication is similar in prokaryotes and eukaryotes, but there are some important differences:
  • E. coli:
    • Genome: 4.6 million bp
    • Single circular chromosome
    • One origin of replication
    • Replication takes ~40 min
    • DNA Pol III and I
    • Primase
    • SSB
  • Human:
    • Genome: 6.0 billion bp
    • 23 pairs of linear chromosomes
    • Thousands of replication origins
    • Replication takes ~8 hr
    • DNA polymerase δ/ε
    • DNA polymerase α
    • Replication protein A: RPA

Special Challenges with Linear Chromosomes

• The presence of free termini can create problems:
  • Potentially vulnerability to exonucleases
  • A replication problem:
    • Replication must proceed 5’-to-3’.
    • It is impossible to cover the 3’-end of the template with DNA.
    • Results in a loss of 10 – 50 nucleotides of DNA / cell cycle.
  • Special properties of chromosome end help to address challenges
    • Ends (telomeres) contain hundreds of repeats of six-nucleotide sequences.
    • The sequence is G-rich (AGGGTT in humans).
    • Allows loop formation to protect telomeric DNA.
  • A dedicated enzyme (telomerase) maintains telomeres by generating chromosomal ends.
    • The protein has an RNA component.
    • It uses the 3’-OH of the single-stranded overhang as a primer and its own RNA template to extend telomeric DNA with reverse transcriptase activity.

Telomeres and Disease

• Dysfunctional telomerase can arise from multiple sources like mutations to RNA in the enzyme, or mutations in telomerase reverse transcriptase (TERT).
• Shorter-than-normal telomeres can lead to disease; rapidly dividing cells are most affected, and shortening is associated with aging.
• Longer-than-normal telomeres can also lead to disease, telomerase is inactive in most cells but is often reactivated in cancer.
• Irrespective of the cause, long telomeres may protect mutations.