Lecture 2

• DNA damage

  Any change from the normal nucleotide sequence and supercoiled double helical state
• Causes of DNA damage

  • Physical and chemical agents in the environment e.g. UV light, free radicals produced during metabolism etc.
  • Errors in DNA replication
• Single Base Changes

  Produces mutations but have no effect on transcription or replication. Eg, chemical modification of bases
• Structural Distortions

  May impede transcription and/or replication, e.g. thymine dimer formation, removal of a base
• Thymine Dimer Formation

  • Cause: Thymine dimers are formed when two adjacent thymines on the same DNA strand become covalently linked due to UV light exposure. This covalent linking results in a cyclobutane structure or a (6-4) photoproduct.
  • Impact: The formation of thymine dimers causes a structural distortion in the DNA helix, which can potentially impede essential cellular processes like transcription and replication.
• DNA Repair Mechanisms (in order of effectiveness)

  • Direct Repair: resolving the problem, reversal or removal of damage
  • Mismatch Repair: detection and repair of mismatched bases
  • Excision Repair: excised and replaced
  • Tolerance Systems: allows DNA replication to process through damaged DNA regions
  • Retrieval Systems (Daughter Strand Gap Repair): recombination
• Photoreactivation

  An example of direct repair where UV-induced intrastrand pyrimidine dimers (usually thymine dimers) are repaired via UV light
• Mismatch Repair

  1. AP endonuclease makes a nick for DNA polymerase to bind

  2. The Mut system is critical in this process

  3. MutS recognizes mismatches/damage and binds to them

  4. MutL binds and stabilizes the complex

  5. MutS-MutL activates MutH, which nicks the newly synthesized strand opposite the nearest methyl group (after replication therefore damage likely on newly synthesized strand)

  6. MutU (Helicase II) unwinds the DNA from the nick towards the mismatch

  7. DNA PolI then degrades and replaces the unwound DNA, and DNA ligase seals the break
• Excision Repair

  1. In E. coli, there are three modes: very short patch (fixes mismatched base pairs), short patch (~20 nucleotides), and long patch (1500-10,000 bps)

  2. These utilise the repair endonuclease encoded by uvrA, uvrB, and uvrC genes

  3. UvrABC endonuclease binds to damaged regions, makes incisions on both sides of the damage, UveD separates the strand before DNA PolI and DNA ligase replace the excised segment
• Tolerance Systems

  • Allows DNA replication to process through damaged DNA regions
  • These are inducible error-prone repair mechanisms
  • Low-fidelity DNA polymerases, known as translesion synthesis polymerases (TSPs), synthesise DNA past damaged bases
  • TSPs are inefficient at replicating undamaged DNA accurately and lack proof-reading ability
  • Examples in E. coli are polymerases IV and V. Humans have 5
  • Human polymerase can bypass the major UV photoproduct very efficiently, usually inserting the correct nucleotides. It is less efficient with most other types of damage
• Retrieval Systems (Daughter Strand Gap Repair)

  This system does not repair damage directly but allows replication to occur successfully, relying on other processes like excision repair to repair the damage afterwards
• SOS Response

  • A system activated in response to severe DNA damage. It leads to the expression of a wide array of genes involved in DNA repair, error-prone DNA replication, and other protective functions
  • This response allows cells to survive and replicate despite the presence of damaged DNA, but it comes at the cost of increasing the likelihood of mutations, contributing to genetic variability and potentially to antimicrobial resistance
• Activation of the SOS Response

  1. The SOS response is controlled by the LexA repressor protein, which binds to specific DNA sequences (LexA boxes) and inhibits the expression of SOS genes under normal conditions

  2. In the presence of DNA damage (e.g., single-stranded DNA), RecA protein binds to the damaged sites, undergoes a conformational change to an active form (RecA*), and facilitates the autocleavage and inactivation of LexA repressor
• General Mechanisms of Transcription Initiation Control

  • Induction: Activates genes as needed.
  • Repression: Deactivates genes when not required.
  • Constitutive Expression: Some genes are always active, unaffected by induction or repression.
  • Operon System: In bacteria and archaea, related genes are grouped into operons and controlled as a single unit, allowing for coordinated regulation.
• Operon Regulation

  • Related genes are often organised into operons, which are transcribed together from a single promoter as a polycistronic mRNA. This organization allows for coordinated regulation of genes with related functions.
  • Repressors: Prevent transcription by binding to DNA near the gene's promoter.
  • Activators (Apoinducers): Facilitate transcription initiation by interacting with DNA.
  • Effectors: Small molecules that toggle regulatory proteins between active and inactive states, influencing gene expression. Inducers switch genes on, while co-repressors switch them off.
• Regulons and Global Regulation

  • Regulons: Collections of operons controlled by a single regulatory protein, managing genes related to a specific function.
  • Global Regulation: Systems managing gene expression across various metabolic functions in response to environmental changes.
• Examples of Regulation in E. coli

  • SOS response: Activates genes for DNA repair.
  • Heat shock: Triggers genes for protein repair and folding.
  • Anaerobic respiration: Manages genes for oxygen-limited metabolism.
  • Catabolite repression: Coordinates sugar metabolism, preferring glucose over other sources.
• Diauxic Growth

  • E. coli shows preference for glucose over lactose, leading to diauxic growth patterns where glucose is consumed first.
  • Catabolite Repression: The presence of glucose inhibits the synthesis of enzymes for metabolizing other sugars, including lactose.
• Lac Operon Components

  • Comprises genes lacZ, lacY, and lacA, involved in lactose metabolism.
  • lacI gene produces the lac repressor that binds to the operator sequence (lacO) to prevent operon transcription in the absence of lactose.
  • Induction by Lactose: Lactose or its metabolites inactivate the repressor, allowing for operon transcription and enzyme production necessary for lactose metabolism.
• Addiction Cassettes

  • used in genetic modification to maintain specific plasmids
  • contain toxin and antitoxin. because antitoxin degrades faster than toxin, toxin will be present after plasmid is removed. Therefore cell without plasmid present will die.