7.2 DNA Damage and Repair Mechanisms

DNA damage is a constant threat to genetic integrity. This section explores various mechanisms cells employ to detect and repair DNA damage, from simple base modifications to complex double-strand breaks.

Understanding these repair processes is crucial for grasping how cells maintain genomic stability. We'll examine key enzymes and pathways involved in fixing different types of DNA damage, highlighting their importance in preventing mutations and disease.

DNA Repair Mechanisms

Base and Nucleotide Excision Repair

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• Base excision repair (BER) removes damaged bases from DNA
  • Initiated by DNA glycosylases recognizing and removing specific damaged bases
  • Creates an apurinic/apyrimidinic (AP) site
  • AP endonuclease cleaves the DNA backbone at the AP site
  • DNA polymerase fills the gap with correct nucleotides
  • DNA ligase seals the nick, completing the repair process
• Nucleotide excision repair (NER) removes bulky DNA lesions
  • Recognizes distortions in the DNA helix caused by damage
  • Excises a short stretch of nucleotides (24-32 base pairs) containing the lesion
  • DNA polymerase fills the gap using the undamaged strand as a template
  • DNA ligase seals the remaining nick
  • Repairs damage caused by UV radiation (cyclobutane pyrimidine dimers)

Mismatch and Double-Strand Break Repair

• Mismatch repair (MMR) corrects base-pairing errors and small insertions/deletions
  • Recognizes mismatched base pairs or small loops in the DNA
  • Excises the newly synthesized strand containing the error
  • Resynthesizes the correct sequence using the template strand
  • Occurs primarily during DNA replication to maintain fidelity
• Double-strand break repair addresses the most severe form of DNA damage
  • Homologous recombination (HR) uses a sister chromatid or homologous chromosome as a template
  • Non-homologous end joining (NHEJ) directly ligates broken ends without a template
  • Both pathways involve multiple steps and specialized enzymes
  • Choice between HR and NHEJ depends on cell cycle stage and availability of a homologous template

Types of DNA Damage

UV-Induced DNA Damage

• Ultraviolet (UV) radiation causes formation of pyrimidine dimers
  • Cyclobutane pyrimidine dimers (CPDs) form between adjacent thymine or cytosine bases
  • (6-4) photoproducts create a link between carbon atoms of adjacent pyrimidines
  • Both types of dimers distort the DNA helix and block replication and transcription
  • Repaired primarily by nucleotide excision repair (NER)
• UV radiation induces indirect DNA damage through reactive oxygen species (ROS)
  • ROS can cause oxidative damage to DNA bases
  • Leads to formation of 8-oxoguanine, a mutagenic lesion

Oxidative and Chemical DNA Damage

• Oxidative damage results from reactive oxygen species (ROS)
  • ROS generated by cellular metabolism or environmental factors
  • Causes oxidation of DNA bases, leading to mutagenic lesions (8-oxoguanine)
  • Can result in single-strand breaks or base modifications
• Chemical agents can cause various types of DNA damage
  • Alkylating agents add alkyl groups to DNA bases (methyl methanesulfonate)
  • Crosslinking agents form covalent bonds between DNA strands (cisplatin)
  • Intercalating agents insert between base pairs, distorting the DNA helix (ethidium bromide)

Enzymes in DNA Repair

DNA Glycosylases and Their Functions

• DNA glycosylases initiate base excision repair (BER)
  • Recognize and remove specific damaged or incorrect bases
  • Create an apurinic/apyrimidinic (AP) site by cleaving the N-glycosidic bond
  • Different glycosylases target specific types of damage (uracil-DNA glycosylase removes uracil)
  • Some glycosylases have associated AP lyase activity to nick the DNA backbone
• Specialized glycosylases address specific types of DNA damage
  • 8-oxoguanine DNA glycosylase (OGG1) removes oxidized guanine bases
  • Thymine DNA glycosylase (TDG) removes thymine from G:T mispairs
  • Alkyladenine DNA glycosylase (AAG) removes alkylated adenine bases

Endonucleases in DNA Repair Pathways

• AP endonucleases cleave the DNA backbone at AP sites
  • APE1 is the primary AP endonuclease in human cells
  • Creates a single-strand break with a 3'-OH and a 5'-deoxyribose phosphate
  • Prepares the site for subsequent steps in base excision repair
• Structure-specific endonucleases function in various repair pathways
  • XPF-ERCC1 and XPG endonucleases make incisions in nucleotide excision repair
  • FEN1 (flap endonuclease 1) removes 5' flaps during Okazaki fragment processing and long-patch BER
  • MUS81-EME1 resolves Holliday junctions in homologous recombination

Double-Strand Break Repair Pathways

Homologous Recombination Mechanism

• Homologous recombination (HR) uses a homologous template for accurate repair
  • Initiated by 5' to 3' resection of DNA ends to create 3' single-stranded overhangs
  • RAD51 protein forms a nucleoprotein filament on the single-stranded DNA
  • Filament invades the homologous template, forming a D-loop structure
  • DNA synthesis extends the invading strand using the template
  • Resolution of the resulting Holliday junction completes the repair process
• HR occurs primarily in S and G2 phases of the cell cycle
  • Requires the presence of a sister chromatid or homologous chromosome
  • Provides high-fidelity repair by using a template
  • Important for repairing complex DNA damage and maintaining genomic stability

Non-Homologous End Joining Process

• Non-homologous end joining (NHEJ) directly ligates broken DNA ends
  • Ku70/Ku80 heterodimer binds to DNA ends and recruits DNA-PKcs
  • End processing enzymes (Artemis) remove damaged nucleotides or create compatible ends
  • DNA polymerases fill in gaps if necessary
  • DNA ligase IV seals the break, completing the repair
• NHEJ can occur throughout the cell cycle
  • Does not require a homologous template
  • Can result in small insertions, deletions, or substitutions at the break site
  • Faster than HR but potentially less accurate
  • Important for repairing double-strand breaks in non-replicating cells (neurons)