Spontaneous and Induced DNA Damage & Repair

Learning Objectives

• Summarize the causes of spontaneous and induced DNA damage that lead to mutations following DNA replication
• Illustrate the molecular mechanisms that repair distinct types of DNA damage.

Mutations

• Mutations can arise spontaneously or be induced by external factors
• Spontaneous mutations occur naturally in all cells
• Induced mutations result from external agents known as mutagens
• Mutations contribute to evolution and disease.

Molecular Basis of Spontaneous Mutations

• Salvador Luria & Max Delbrück (1943) researched the molecular basis of spontaneous mutations.
• Mutations can occur spontaneously due to cellular processes acting on DNA.
• E. coli, infected by T₁ phages, developed resistance, which was heritable and resulted from mutation.
• Mutations can be spontaneous or induced by infection

Molecular Basis of Spontaneous Mutations: Fluctuation Test

• The fluctuation test was designed to determine if mutations occurred before (spontaneous) or after exposure (induced) to a selective agent (phage).
• The fluctuation test was conducted through two treatment groups.
• If mutations were induced by the phage, mutations occur later only after phage exposure and minimal fluctuation is expected in the number of resistant colonies

Molecular Basis of Spontaneous Mutations: Spontaneous vs Induced Mutation

• If mutations are spontaneous, mutations occur during each generation regardless of phage exposure
• If mutations are spontaneous, early mutations will lead to many resistant descendants with larger fluctuations in numbers of resistant colonies
• All plates from aliquots had similar numbers of resistant colonies (14-26)
• Plates from individual cultures had larger variation in the number of resistant colonies (0-107)

Mechanisms of Spontaneous Mutations

• Spontaneous mutations arise from:
  • DNA replication errors
  • DNA damage from the cellular environment
  • Insertion of transposable elements.

Errors in DNA Replication

• DNA replication is generally accurate but mistakes do happen.
• Mismatched base pairs can lead to transition or transversion substitutions
• Mismatching is caused by tautomerization and ionization of bases

Errors in DNA Replication: Tautomers and Base Pairing

• Each base in DNA can exist in several forms called tautomers, which are structural isomers differing in atom and bond positions.
• Keto form is normally present while enol form is rarer.
• The enol or imine form of a base may pair with the wrong base.

Errors in DNA Replication: Ionization and Wobble Base Pairing

• Ionization occurs when a base acquires a positive or negative charge.
• Ionization is due to interactions with water which resemble a wobble base pair.

Errors in DNA Replication: Slippage

• Errors in DNA replication can also cause indels, involving insertions or deletions.
• Loops in single-stranded DNA are stabilized by "slipped mispairing" of repeated sequences, resulting in replication slippage.

The Cellular Environment: Spontaneous Mutations

• Mutations can arise due to DNA damage from water and reactive oxygen species (ROS).
• Reactions can cause depurination and deamination.
• ROS reactions can cause several types of DNA damage.

The Cellular Environment: Depurination

• Depurination is the loss of a purine base (A or G).
• Depurination is due to hydrolysis of the glycosidic bond between the base and deoxyribose.
• The nitrogenous base is lost during depurination, but sugar-phosphate backbone stays intact.

The Cellular Environment: Deamination

• Deamination is the removal of an amino group from cytosine, adenine, or guanine.

The Cellular Environment: Oxidative Damage

• Oxidative damage is caused by reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals.
• It causes many types of modifications, with over 100 identified.

Molecular Basis of Induced Mutations

• Some mutations are caused by agents present in external environment equals induced mutations
• Mutagens can be present in air, food, water, etc.
• Induced mutations can be contributed by chemical (ROS, base analogs, alkylating agents, intercalating agents) and physical (ultraviolet light, ionizing radiation) sources.

Molecular Basis of Induced Mutations by Mutagens

• Mutagens induce mutations by at least three mechanisms:
  • Replacing a base in DNA
  • Altering a base so that it mispairs
  • Damaging a base so that it can no longer pair with any base

Base Modification by Alkylating Agents

• Alkylation is the addition of an alkyl group (e.g. CH3) or an ethyl group (C2H5) to a nucleotide base.
• Prevent normal base pairing.

Base Damage by Bulky Adducts.

• Bulky adducts attach to nucleotides to induce mutations.
• Aflatoxin B₁ attaches to guanine, breaks glycosidic bond, leads to apurinic site.

Incorporation of Base Analogs

• Some compounds similar to nitrogenous bases can take their place as a base analog
• To cause a mutation, analog must mispair more often that base it replaces

Binding of Intercalating Agents

• Intercalating agents mimic base pairs and slip between (intercalate) nitrogenous bases in the double helix
• May fool DNA polymerase into inserting extra bases or skipping certain bases

Base Damage by UV Light

• UV light can cause pyrimidine dimers, which involves covalent bonds between pyrimidine bases on the same strand.
• These bases cannot form proper base pairs, can block replication (and/or transcription) and lead to inclusions of the wrong base

Base Damage and Modification: Ionizing Radiation

• Ionizing radiation can cause damage by generating ROS.
• It can directly damage DNA by breaking glycosidic bonds, forming abasic sites, which causes single strand breaks, and double strand breaks (DSBs).

DNA Repair Mechanisms

• All organisms use a variety of mechanisms to repair DNA damage
• The major pathways include:
  • Base excision repair (BER)
  • Nucleotide excision repair (NER)
  • Mismatch repair (MMR)
  • Translesion synthesis (TLS)
  • Homologous recombination (HR)
  • Nonhomologous end joining (NHEJ)

Direct Repair of Damaged DNA

• The easiest way to repair damage is to directly reverse it to regenerate a normal base.
• Pyrimidine dimers are repaired by CPD photolyase
• Alkylation is repaired by MGMT.

Base Excision Repair (BER)

• BER corrects small base lesions by removing the damaged base and inserting a normal one.
• This repair works with damage caused by alkylation, oxidation, and deamination.

Base Excision Repair (BER) steps

• Damage is detected by DNA glycosylase, which cleaves the glycosidic bond
• AP endonuclease "nicks" the damage strand upstream and another enzyme removes the sugar phosphate backbone
• DNA polymerase inserts new nucleotides
• Ends are joined with DNA ligase.

Nucleotide Excision Repair (NER)

• NER can repair damage from bulky adducts, pyrimidine dimers, or damage to multiple bases.
• There are six common steps.

Nucleotide Excision Repair (NER) Steps

• Damage detection
  • Binding of specific proteins or stalling of RNA polymerase detect damaged nucleotides
• Strand separation
  • A helicase enzyme separates the DNA strands
• Incision (cleavage)
  • Endonucleases cleave phosphodiester bonds upstream and downstream of the damage site
• Excision
  • Several enzymes remove the damaged stretch of DNA
• Synthesis (polymerization)
  • DNA polymerase replaces damaged nucleotides
• Ligation
  • Ends are joined by DNA ligase

Mismatch Repair (MMR)

• DNA polymerase has proofreading activity and reduces the error rate in replication to about 1 error in 10 million base pairs.
• The MMR pathway corrects remaining errors and reduces the rate to 1 in 1 billion.

Mismatch Repair (MMR) Steps

• Detection
  • Mismatches in newly replicated DNA are detected by the MutS protein
  • MutS binds to the mismatch, and recruits MutL and MutH
• Incision
  • MutH cuts the newly synthesized strand with the incorrect base
  • Identifies new/old strands via adenine methylation at GATC sequences, and new strand is unmethylated
• Strand separation and excision
  • Strands are separated via helicase
  • Damaged portion is removed via exonuclease
• Synthesis and Ligation
  • New DNA is synthesized via DNA polymerase
  • Ends are joined together by DNA ligase

Translesion Synthesis (TLS)

• Lesions that stall/stop DNA replication can cause severe consequences including cell death.
• A variety of DNA polymerases are used to replicate past lesions to allow for complete genome duplication = translesion synthesis.
• Provides additional time for DNA to be repaired.

Translesion Synthesis Process

• TLS is initiated by a stalled DNA polymerase
  • Occurs at apurinic sites, bulky adducts, and pyrimidine dimers
• Triggers recruitment of a special TLS polymerase
• TLS polymerase synthesizes past the lesion
• Replicative polymerase replaces the TLS polymerase and continues with synthesis once extension has passed the lesion

Translesion Synthesis (TLS): TLS polymerases

• TLS polymerases differ from other DNA (replicative) polymerases in three important ways:
  • Replicative pol stall because damaged bases do not fit in active site
    • TLS pol have a much larger active site to accommodate damaged bases
  • TLS pol lack proofreading ability and can be error prone
  • TLS pol can only add a few nucleotides before falling of DNA template

Repair of Double-Stranded Breaks (DSBs)

• Many DNA repair mechanisms rely on complementary base pairing to produce error-free repairs.
• However, it is not possible to base pair with DSBs.
• If DSBs are left unrepaired, can have severe consequences (cancers, cell death, etc.).
• Eukaryotes use two primary pathways for double-stranded breaks

Repair of Double-Stranded Breaks Pathways

• Nonhomologous end joining (NHEJ)= joins DNA ends independent of sequence complementarity
  • Can be prone to incorporating errors
  • Functions in both dividing and non-dividing cells
• Homologous recombination (HR)= complementary sequence on a homologous chromosome used as a template to extend DNA past break point
  • Primarily occurs in S and G₂ phases of the cell cycle, restricted to actively dividing cells