07-Biochemistry-Lecture7 | Week 4 - Lecture 2

LECTURE 7: DNA REPLICATION

DNA METABOLISM

  • Definition: DNA metabolism refers to a set of tightly regulated processes that maintain the integrity, stability, and expression of genetic information.

  • Overview of Processes:

    • DNA Replication: A new copy of DNA is synthesized before each cell division with high fidelity.

    • DNA Repair: Errors arising during or after DNA synthesis are constantly checked for and repaired.

    • DNA Recombination: Segments of DNA are rearranged either within one chromosome or between two DNA molecules, providing genetic diversity to offspring.

  • Summary: DNA serves as the substrate that encodes its own metabolism.

OUTLINE OF REPLICATION

  • Main Topics:

    • General principles of replication

    • Structure and reaction of polymerases

    • Key concepts: processivity, error rates, proofreading

  • Stages of Replication:

    • Initiation, Elongation, Termination

  • Comparison: Prokaryotic vs Eukaryotic processes

DNA REPLICATION FEATURES

  • Three Fundamental Rules of Replication:

    1. Semi-Conservative: Each new DNA molecule consists of one old strand and one new strand.

    2. Bi-Directional: Replication begins at an origin and proceeds in two directions.

    3. Directionality: Synthesis of new DNA occurs in the 5’ → 3’ direction and is semi-discontinuous.

THE MESELSON-STAHL EXPERIMENT

  • Purpose: Demonstrated that DNA replication is semiconservative.

  • Methodology:

    • Cells were grown in a medium containing heavy nitrogen (15N) so that their DNA contained only 15N.

    • After transferring cells to light nitrogen (14N) for one generation, DNA was isolated and centrifuged in a CsCl gradient, yielding a hybrid DNA band indicative of semiconservative replication.

  • Results: The first generation showed a single hybrid band; the second generation showed one hybrid and one band of lighter 14N DNA, confirming semiconservative replication.

REPLICATION OF CIRCULAR DNA

  • Bidirectional Nature: DNA replication begins at an origin and usually progresses bidirectionally.

  • Visualization of Bidirectional Replication:

    • Stages are visualized by electron microscopy, producing a theta (θ) structure as both strands are replicated simultaneously.

    • Electron micrographs illustrate replication of plasmid DNA from a single origin.

SEMI-DISCONTINUOUS SYNTHESIS

  • Replicative Strands at the Fork:

    • A new strand is always synthesized in the 5’ → 3’ direction, reading the template in the opposite direction (3’ → 5’).

    • Leading Strand: Continuously synthesized in the direction of the replication fork.

    • Lagging Strand: Synthesized discontinuously in short pieces (Okazaki fragments) in the opposite direction to the fork's movement.

    • Okazaki Fragment Lengths: In bacteria, 1000 to 2000 nucleotides; in eukaryotes, 150 to 200 nucleotides.

STRUCTURAL FEATURES OF DNA POLYMERASES

  • Core Structure: A DNA polymerase has a pocket with two regions:

    • Insertion Site: Where the incoming nucleotide binds.

    • Post-Insertion Site: Where the newly made base pair resides after the polymerase moves forward.

  • Shape: Most DNA polymerases resemble a human hand that wraps around the active site, as seen in DNA Polymerase I from Thermus aquaticus.

DNA ELONGATION CHEMISTRY

  • Template: Parental DNA strand serves as a template for elongation.

  • Substrates: Nucleoside triphosphates (dNTPs) are used as substrates in strand synthesis.

  • Mechanism of Nucleotide Addition: The nucleophilic OH group at the 3’ (the primer) end of the growing chain attacks the α-phosphate of the incoming trinucleotide.

    • This 3’-OH is REQUIRED

    • The 3’-OH is made a more powerful nucleophile by nearby Mg2+ ions

    • Pyrophosphate is a good leaving group

  • Reaction Representation:

MECHANISM OF ACTION OF DNA POLYMERASES

  • Catalytic Mechanism: Involves two Mg²⁺ ions and three highly conserved Asp residues.

  • Function of Mg²⁺ Ions: Facilitate attack by the 3’-hydroxyl group on the α phosphate and displace pyrophosphate (PPi).

  • Processivity of Polymerase: Refers to the number of dNTPs added before the polymerase dissociates from the template.

  • Requirements for Activity: Template, primer, and dNTPs are required.

FEATURES AND IMPORTANCE OF A PRIMER

  • Primer: A short strand complementary to the template that serves as a starting point for DNA synthesis.

    • Contains a free 3’-OH to begin the first DNA polymerase-catalyzed reaction

    • Can be made of DNA or RNA

  • 3’-OH Group: Essential for DNA polymerase-catalyzed reactions, allowing the addition of nucleotides to the growing strand.

  • Base-Pairing: Each incoming nucleotide is selected based on base-pairing with the template strand. This insertion occurs in the insertion site.

  • The newly formed base pair migrates to the post-insertion site to make the active site available to the next pair to be formed.

CONTRIBUTION OF BASE PAIR GEOMETRY TO REPLICATION FIDELITY

  • Base Pair Geometry: Proper A=T and G≡C pair geometries allow them to fit into the polymerase active site. Incorrect pair geometries can be excluded from the active site, reducing errors.

HIGH FIDELITY OF DNA SYNTHESIS

  • Error Rates of DNA Synthesis:

    • Geometry of base pairing & proofreading accounts for high fidelity

    • DNA polymerase active site excludes base pairs with incorrect geometry, & it also has a proofreading activity.

      • DNA polymerase adds nucleotides with an error rate of 1 in 10⁵ (because of tautomerism → H-bonding to incorrect partner).

      • Proofreading still does not correct errors every 1/10² bases

        • Proofreading (3’ → 5’ exonucleolytic): 1 in 10².

      • Strand-directed mismatch repair: 1 in 10³.

      • Combined Rate: Approximately 1 error in 10¹⁰ bases replicated.

ERRORS DURING SYNTHESIS AND CORRECTION

  • Proofreading Mechanism: Most DNA polymerases have a 3’ → 5’ exonuclease activity that corrects mismatched base pairs.

  • Translocation Process: The enzyme is inhibited from moving to the next nucleotide until the incorrect nucleotide is removed.

  • Error Correction Process: The enzyme slides the DNA backward into the exonuclease site to correct mistakes.

ERROR CORRECTION BY 3’→ 5’ EXONUCLEASE ACTIVITY OF DNA POLYMERASE I

  • The exonuclease activity is located behind the polymerase activity as the enzyme is oriented in its movement along the DNA.

  • A mismatched base impedes translocation of DNA polymerase I to the next site.

  • The DNA bound to the enzyme slides backward into the exonuclease site, and the enzyme corrects the mistake with its 3’→5’ exonuclease activity.

  • The enzyme then resumes its polymerase activity in the 5’→3’ direction.

  • Proofreading activity of Pol I improves the accuracy of polymerization by 102 to 103 fold.

KEY TERMINOLOGY FOR POLYMERASES

  • Fidelity: The error rate in the 5’ to 3’ synthesis.

  • Proofreading: The capability to correct synthesis errors.

  • Processivity: The number of nucleotides added before dissociation from the template.

  • Rate: The speed of nucleotide addition.

DNA POLYMERASE DIVERSITY IN E. COLI

  • DNA Polymerase I: Abundant but slow (10-20 nucleotides/sec) with low processivity (3-200 bases) → Mainly involved in clean-up.

  • DNA Polymerase III: Principal replication polymerase with high rate and processivity.

  • DNA Polymerases II, IV, and V: Involved mainly in DNA repair.

COMPARISON OF E. COLI DNA POLYMERASES

  • Role of Pol II, IV, and V: Primarily focused on DNA repair activities.

NICK TRANSLATION AND MULTI-FUNCTIONAL DNA POLYMERASE I

  • 5’ → 3’ Exonuclease Activity: DNA Polymerase I also has this activity, which removes nucleotides in the direction of movement, facilitating nick translation.

    • DNA polymerase I degrades the DNA ahead of the enzyme (in 5’ → 3’ direction), thus removing nucleotides on its path.

  • Klenow Fragment: A distinct domain that separates from DNA Polymerase I, but lacks the 5’ → 3’ exonuclease activity.

    • comprises the 5’→3’ polymerase activity & the 3’→5’ proofreading exonuclease activity.

NICK TRANSLATION MECHANISM

  • Mechanism Description: Movement of the nick along with the enzyme due to its polymerase and exonuclease activities allows for removal of RNA primers during replication.

  • Bacterial DNA polymerase I has three domains, which catalyze: DNA polymerase activity, 5’→3’ exonuclease activity, and 3’→5’ exonuclease activity

  • The 5’→3’ exonuclease domain is positioned in front of the enzyme as it moves along DNA.

  • DNA polymerase I promotes a reaction called nick translation by:

    • Degrading the DNA strand ahead of the enzyme

    • Synthesizing a new DNA strand behind the enzyme

  • During nick translation, a break (nick) in the DNA is effectively moved along with the enzyme.

  • Nick translation plays a role in: DNA repair & Removal of RNA primers during DNA replication.

  • A nick remains after DNA polymerase I dissociates. This remaining nick is later sealed by another enzyme.

EUKARYOTIC DNA POLYMERASES

  • Diversity in Eukaryotes: 15 DNA polymerases present in eukaryotes, such as:

    • DNA Pol α: Extends primers initiated by primase.

    • DNA Pol δ: Used in the lagging strand.

    • DNA Pol ε: Functions in the leading strand.

    • DNA Pol β and others: Used for repair purposes.

    • DNA Pol γ: Responsible for mitochondrial DNA replication.

INITIATION OF REPLICATION

PROKARYOTIC INITIATION

  • Single Origin (oriC): Recognized by DnaA.

  • DnaA Role: Induces melting at DUE (DNA Unwinding Element) due to high AT content.

  • Other Proteins Required: DnaB (helicase), DnaC (helicase loader), clamp loader for polymerase, and SSB (single-strand binding protein).

  • Primase makes first primers.

  • Pol III starts synthesis of DNA

  • Only 1 replicstion/cylce:

    • DnaA ATP/ADP hydrolysis

    • SeqA sequestration

EUKARYOTIC INITIATION

  • Multiple Origins (1000): Recognized by the origin of replication complex (ORC).

  • Helicase Loading: MCM2-7 opens the site, cyclin signaling induces changes, RFC acts as a clamp loader and RPA stabilizes ssDNA.

  • Primase & DNA pol α make first primers.

  • DNA Pol ε & δ start synthesis of DNA.

  • Only 1 replication/cycle: sites licensed before S phase.

DNA POLYMERASE III (POL III)

  • Architecture: Composed of three core domains and a five-subunit clamp-loading complex. Maintains high processivity and interaction with helicase.

  • Functionality: Provides effective coordination during lagging and leading strand synthesis, crucial for overall replication speed and fidelity.

ELONGATION STAGE OF REPLICATION

PROKARYOTIC ELONGATION

  • Process Complexity: Involves multiple proteins including DNA polymerases, primase, and helicase.

  • Speed: Fast → Approximately 500 bp/s per polymerase, with a total replication time of about 40 minutes for a typical bacterial genome.

  • Primer Processing: RNA primers are removed by DNA Polymerase I.

EUKARYOTIC ELONGATION

  • Replication Duration: Slower than prokaryotic elongation, taking 6-8 hours to replicate the human genome.

  • Speed: Approximately 50 bp/s per polymerase.

  • Primer Removal: Carried out predominantly by Dna2 and FEN1.

COMPLEXITY OF DNA REPLICATION ENZYMES

  • E. coli requires over 20 enzymes and proteins for DNA replication

  • A set of proteins involved in replication, called the replisome, includes:

    • DNA-binding proteins to stabilize separated strands

    • Helicases (use ATP to unwind DNA strands)

    • Primases (make RNA primer)

    • DNA polymerases I and III (primer removal and DNA elongation)

    • DNA ligases to seal nicks

    • DNA gyrases (also called DNA topoisomerases II; relieve the stress caused by unwinding)

  • Eukaryotes require far more proteins, but similar activities

TOPOISOMERASE FUNCTION

  • Winding Problem: During replication movement, the ahead parent DNA helix must rotate to prevent overwinding, which could halt replication.

  • Role of Topoisomerases: Generating temporary single-strand breaks that relieve tension by allowing strand rotation, thus enabling continuous replication without halting.

LAGGING STRAND SYNTHESIS STEPS

  • Primer Removal and Replacement: Conducted by DNA polymerase I using its 5’ → 3’ exonuclease activity; remaining nicks are sealed by DNA ligase.

  • Nick Sealing Mechanism: 5’-PO4 must be activated before nucleophilic attack by 3’-OH, resulting in complete strand integrity.

GENERAL FEATURES OF THE REPLICATION FORK

  • The DNA duplex is unwound by: DNA gyrase & Helicase

  • Single-stranded regions are coated by SSB (single-strand binding proteins)

  • Primase periodically adds RNA primers.

  • The two sliding clamps of DNA polymerase III replicate the strands.

  • DNA polymerase I and DNA ligase act downstream on the lagging strand to:

    • Remove RNA primers and replace them with DNA (Pol I)

    • Ligate (join) the Okazaki fragments (Ligase)

TERMNATION OF REPLICATION

PROKARYOTIC TERMINATION

  • Usage of Ter Sequences: Bind Tus protein to signal replication ends; resolution of catenated circles occurs via topoisomerases.

EUKARYOTIC TERMINATION

  • Mechanism: Replicative forks meet and fuse, with unreplicated ends filled by telomerase, maintaining genomic stability through generations.

Termination of replication in E. coli

  • The Ter sequences (TerA through TerF) function as a trap for the replication fork.

  • Ter sequences bind Tus proteins (Terminus Utilization Sequence).

  • Topoisomerases are required in replication termination.

  • When opposing replication forks meet:

    • Replication leaves the completed chromosomes joined as catenanes (topologically interlinked circles).

    • Because they are covalently closed, they cannot be separated without topoisomerases.

  • In E. coli, a type II topoisomerase (DNA topoisomerase IV):

    • Plays the primary role in separating catenated chromosomes

    • Transiently breaks both DNA strands of one chromosome

    • Allows the other chromosome to pass through the break