Week 9 S - DNA Damage and Repair Notes

DNA Damage and Repair

Introduction

• The 2015 Nobel Prize in Chemistry was awarded to Tomas Lindahl, Paul Modrich, and Aziz Sancar for their mechanistic studies of DNA repair.

• DNA damage and repair are essential topics in genetics.

Learning Outcomes

• Describe the types of DNA damage that can occur, their sources, and their specific effects.

• Understand and discuss the DNA repair mechanisms specific to different types of DNA damage.

• Discuss the importance of DNA damage-repair mechanisms.

• Uncover the links between DNA damage and disease.

What is DNA Damage?

• DNA is the repository of genetic information, and its integrity and stability are essential for life.

• DNA is not inert; it is subject to assault from the environment and from endogenous sources.

• If not repaired, DNA damage will lead to mutation and possibly disease.

• DNA damage is an abnormal chemical structure in DNA, while a mutation is a change in the sequence of standard base pairs.

• DNA damages can cause changes in the structure of the genetic material, preventing the replication mechanism from functioning properly.

Sources of DNA Damage

• Spontaneous:

  • Spontaneous replication errors: DNA/genetic insults caused by the process of DNA replication during cell division; replication is prone to error.

  • Spontaneous mutation rate in humans: $1 \times 10^{-4}$ to $1 \times 10^{-6}$ mutations per gamete for a given gene.

  • Spontaneous Chemical Changes

• Chemically induced:

  • Environmental agents that significantly increase the rate of mutations are called mutagens (base analogs, alkylating agents, deaminating chemicals, oxidative agents, intercalating agents).

• Radiation:

  • Environmental-induced DNA damage and links to disease:

    • Skin cancer caused by excessive exposure to UV radiation in sunlight.

    • Damage caused by tobacco smoke, which can lead to mutations in lung cells and subsequent lung cancer.

Spontaneous Replication Errors

• Tautomeric shifts

• Mispairing due to other structures

• Incorporation of wrong nucleotides

• Deletions and insertions

Tautomeric Shifts

• Tautomeric shifts involve the relocation of a proton, leading to altered base-pairing properties.

Spontaneous Chemical Changes

• Depurination:

  • Loss of a purine base.

  • During replication, the apurinic site cannot provide a template for a complementary base.

  • A nucleotide with an incorrect base (most often A) is incorporated into the newly synthesized strand.

  • At the next round of replication, this incorrectly incorporated base will be used as a template, leading to a permanent mutation.

• Deamination:

  • Loss of an amino group.

  • Deamination of cytosine converts it to uracil.

  • Deamination of 5-methylcytosine (5mC) converts it to thymine.

Chemically Induced Mutations

• Base analogs:

  • Chemicals with structures similar to normal bases that can be incorporated into DNA.

  • Example: 5-Bromouracil, which can mispair with guanine.

  • Incorporation of bromouracil followed by mispairing leads to a TA → CG transition mutation.

• Alkylating agents:

  • Donate alkyl groups to bases.

  • Examples: Ethylmethylsulfonate (EMS), Mustard gas.

• Deamination:

  • Nitrous acid.

• Hydroxylamine:

  • Adds hydroxyl group.

Chemically Induced Mutations (cont.)

• Oxidative reaction:

  • Superoxide radicals.

  • Hydrogen peroxide.

• Intercalating agents:

  • Proflavin, acridine orange, and ethidium bromide.

  • Insert themselves between adjacent bases in DNA, distorting the structure.

Radiation-Induced Changes

• Ionizing radiation (X-rays, gamma rays, alpha and beta particles, and neutrons):

  • DNA breaks, particularly double-strand breaks (DSBs).

  • Generation of reactive oxygen species (ROS) causing abasic sites and single-strand breaks (SSBs).

• UV radiation:

  • Two of the most abundant mutagenic and cytotoxic DNA lesions include:

    • Cyclobutane-pyrimidine dimers (CPDs).

    • 6-4 photoproducts (6-4PPs) and their Dewar valence isomers.

Transition and Transversion

• Transition:

  • Conversion of a purine to another purine base or a pyrimidine to another pyrimidine base.

• Transversion:

  • Conversion of a purine into a pyrimidine or vice versa.

Why is DNA Repair Important?

• Most DNA damages can undergo DNA repair, but such repair is not 100% efficient.

• Unrepaired DNA damages accumulate in non-replicating cells (e.g., brain or muscle cells in adult mammals) and can cause aging.

• In replicating cells (e.g., cells lining the colon), errors occur upon replication past damages in the template strand of DNA or during repair of DNA damages.

• These errors can give rise to mutations or epigenetic alterations that can be replicated and passed on to subsequent cell generations.

• These alterations can change gene function or regulation of gene expression and possibly contribute to progression to cancer.

DNA Lesions and Their Repair Pathways in Eukaryotes

• Different damaging agents cause different types of DNA lesions, which are then repaired by specific pathways.

• UV-light:

  • 6-4 photoproduct.

  • Cyclobutane pyrimidine dimer, repaired by Nucleotide excision repair (NER)

• Polycyclic aromatic hydrocarbons:

  • Bulky adduct, repaired by Nucleotide excision repair (NER)

• X-rays:

  • Abasic site, repaired by Base excision repair (BER)

• Oxygen radicals:

  • Oxidised, deaminated and alkylated bases, repaired by Base excision repair (BER)

• Alkylating agents:

  • Oxidised, deaminated and alkylated bases, repaired by Base excision repair (BER)

• Spontaneous lonizing radiation:

  • DNA single-strand break (SSB), repaired by Base excision repair (BER)

• X-rays and Anti-cancer agents:

  • DNA double-strand break (DSB), repaired by Double strand break repair (DSBR): Homologous recombination (HR) and Non-homologous end-joining (NHEJ)

• Replication and recombination errors:

  • Base mismatches, Insertion, and Deletion, repaired by Mismatch repair (MMR)

DNA Repair Mechanisms

• Summary of common DNA repair mechanisms:

  • Mismatch repair: Replication errors, including mispaired bases and strand slippage.

  • Direct repair: Pyrimidine dimers; other specific types of alterations.

  • Base excision repair: Abnormal bases, modified bases, and pyrimidine dimers.

  • Nucleotide excision repair: DNA damage that distorts the double helix, including abnormal bases, modified bases, and pyrimidine dimers.

  • Homologous recombination: Double-strand breaks.

  • Nonhomologous end joining: Double-strand breaks.

Repair DNA Polymerases

• Different DNA polymerases are involved in different repair pathways.

  • Pol α: RNA and/or DNA primers

  • Pol β: Base-excision repair

  • Poly: Mitochondrial DNA replication and repair

  • Pol δ: Lagging-strand synthesis, DNA repair

  • Pol ε: Leading-strand synthesis

• Y family polymerases (e.g., Pol η, Pol ι, Pol κ) are involved in bypass synthesis of DNA lesions.

  • n (eta): Bypass UV lesions

  • ι (iota): Bypass synthesis

  • K(kappa): Bypass synthesis

• X family polymerases (e.g., Pol λ, Pol μ) are involved in base-excision repair and NHEJ.

  • λ(lambda): Base-excision repair, NHEJ

  • μ (mu): NHEJ

DNA Repair Types

• Direct repair: Does not replace altered nucleotides but restores their original correct structure.

  • Photoreactivation uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers (CPDs) formed by ultraviolet light.

  • Alkyltransferase removes methyl or ethyl groups from alkylated guanine residues.

  • $6-4PP$: pyrimidine 6–4 pyrimidone photoproduct

  • $CPD$: cyclobutane pyrimidine dimer

Photoreactivation Repair

• Uses a white light-dependent enzyme to split cyclobutane pyrimidine dimers formed by ultraviolet light.

Excision Repair

• One strand of DNA is directly excised and then replaced by resynthesis using the complementary strand as a template.

Mismatch Repair (MMR)

• Corrects mispaired bases immediately following replication.

• Preferentially corrects the sequence of the daughter strand by distinguishing it from the parental strand based on their states of methylation.

Excision Repair

• Base excision repair (BER):

  • Recognizes damage to single bases, such as deamination or alkylation.

  • Repairs the nucleotide alone (short-patch repair) or replaces 2–10 nucleotides (long-patch repair).

• Nucleotide excision repair (NER):

  • Recognizes bulky lesions in DNA (such as UV-induced pyrimidine dimers).

  • Divided into two major subpathways:

    • Transcription-coupled repair (TC-NER): Repairs damage in the transcribed strand of active genes.

    • Global genome repair (GG-NER): Repairs damage anywhere in the genome.

Base Excision Repair

• Each DNA glycosylase recognizes and removes a specific type of damaged base, producing an apurinic or apyrimidinic site (AP site).

• AP endonuclease cleaves the phosphodiester bond on the 5' side of the AP site.

• DNA polymerase adds new nucleotides to the exposed 3'-OH group.

• The nick in the sugar-phosphate backbone is sealed by DNA ligase, restoring the original sequence.

Nucleotide Excision Repair

• Nucleotide excision repair removes bulky DNA lesions that distort the DNA double helix

• Global genome repair (GG-NER) recognizes damage anywhere in the genome.

• Transcriptionally active genes are preferentially repaired via transcription-coupled repair (TC-NER).

• GG-NER: XPC detects damage

• TC-NER: RNA polymerase stalls, recruiting CSB and CSA proteins.

• TFIIH provides the link to a complex of repair enzymes.

Double-Strand (ds) DNA Break Repair

• Physiologic double-strand DNA breaks:

  • V(D)J recombination breaks (RAG1,2).

  • Class switch breaks (AID/UNG/APE).

• Pathologic double-strand DNA breaks:

  • Ionizing radiation.

  • Oxidative free radicals.

  • Replication across a nick.

  • Inadvertent enzyme action at fragile sites.

  • Topoisomerase failures.

  • Mechanical stress.

• Nonhomologous DNA end joining (NHEJ):

  • Ku70/86, DNA-PKcs, Artemis, pol u&A, XRCC4, ligase IV, XLF/Cernunnos.

  • Occurs in the entire cell cycle

• Homology-directed repair (HR & SSA):

  • RAD50, MRE 11, Nbs1 (MRN); RAD51(B,C,D), XRCC2, XRCC3, RAD52, RAD54B, BRCA2, and other proteins

  • Occurs in Late S, and G2 phases.

Nonhomologous End Joining

• Repair of double-strand breaks when homologous sequence is not available occurs through a nonhomologous end joining (NHEJ) reaction.

• The NHEJ pathway can ligate blunt ends of duplex DNA.

• Mutations in double-strand break repair pathways cause human diseases.

Homologous Recombination Repair

• The single strand of another intact DNA duplex is used to replace the gap.

• The damaged sequence is removed and resynthesized.

• The RAD51 group of genes is required for recombination-repair.

• The MRX (yeast) or MRN (mammals) complex is required to form a single-stranded overhang at each DNA end.

• The MRN complex, required for 5’ end resection, also serves as a DNA bridge to prevent broken ends from separating.

DNA Damage Repair Requires Chromatin Remodeling

• Both histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin.

• H2AX phosphorylation is a conserved DSB-dependent modification that recruits chromatin modifying activities and facilitates assembly of repair factors.

DNA Damage Repair Linked to Genetic Disorders

• Different DNA damage types and repair system defects are linked to various genetic disorders.

  • Single strand breaks (SSBs): ataxia oculomotor apraxia 1 (AOA1) and Spinocerebellar ataxia with axonal neuropathy 1 (SCAN1)

  • Various bulky lesions (CPDs and 6-4PPs): Xeroderma pigmentosum (XP) and Combined Xeroderma pigmentosum + Cockayne syndrome and Trichothiodystrophy (TTD)

  • DSBs: Ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS)

  • Interstrand crosslinks (ICLS): Fanconi anemia (FA)

Consequences of DNA Mutations

• Wild type (normal): Normal DNA sequence and protein production.

• Missense mutation: Amino acid change at a specific position.

• Silent mutation: No change in amino acid sequence.

• Nonsense mutation: Premature stop codon, resulting in a truncated protein.

• Frame-shift mutation: Insertion or deletion of bases, leading to a completely different amino acid sequence downstream of the mutation.

DNA Damage: Summary

• DNA damage types and their sources.

• DNA repair types specific for DNA damage types.

• Link between DNA damage and disease.