DNA Damage, Repair, and Homologous Recombination (Chapter 17 Study Notes)

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DNA Damage and Repair

Introduction to DNA Damage and Repair

DNA integrity is essential for the faithful transmission of genetic information to daughter cells. Both spontaneous and environmentally induced alterations, known as mutations, must be repaired to maintain cellular function and prevent disease.

DNA mutations can arise spontaneously or due to environmental agents.
Repair mechanisms are crucial for correcting these alterations.

Spontaneous Mutations During Replication

Several types of mutations can occur during DNA replication, often due to errors in base pairing or DNA structure.

Spontaneous mispairing of bases due to transient formation of tautomers.
Slippage during replication, especially in repetitive DNA regions.
Spontaneous damage to individual bases.

DNA Tautomers

Tautomers are rare, alternate resonance structures of nitrogenous bases that can lead to nonstandard base pairing, known as a tautomeric shift.

Mispairing due to tautomers is the most common form of spontaneous replication error.
Results in daughter strands with incorrect bases.
Example: A tautomeric shift in cytosine can cause it to pair with adenine instead of guanine.

Trinucleotide Repeats

Regions of repetitive DNA, such as trinucleotide repeats, are susceptible to strand slippage during replication, leading to repeat expansion and genetic disorders.

DNA polymerase may replicate a short stretch of DNA twice.
Associated with diseases like Huntington's Disease.

Disease	Repeat Sequence	Normal Repeat Number	Disease Repeat Number	Primary Symptoms
Fragile X syndrome	CGG	6–50	200–2000	Mental retardation
Huntington's disease	CAG	6–37	38–121	Muscle wasting
Myotonic dystrophy type 1	CTG	5–37	50–2000	Muscle weakness
Spinocerebellar ataxia type 1	CAG	6–39	40–83	Loss of motor coordination

Depurination and Deamination

Chemical modifications of DNA bases can occur spontaneously, leading to mutations if unrepaired.

Depurination: Loss of a purine base (adenine or guanine).
Deamination: Removal of an amino group from a base (e.g., cytosine to uracil).
Thousands of depurinations and about 100 deaminations occur daily in human cells.
Unrepaired changes can alter the DNA sequence.

Mutagen-Induced DNA Damage

Mutagens and Their Effects

Mutagens are agents that cause DNA damage, leading to mutations. They can be chemical or physical (radiation), or biological (mobile genetic elements).

Chemicals: Base analogues, base-modifying agents, intercalating agents.
Radiation: Ultraviolet and ionizing radiation.
Mobile genetic elements: Viruses and transposons.

DNA Damage by Chemical Mutagens

Base analogues: Resemble normal bases and are incorporated into DNA (e.g., 5-bromodeoxyuridine, BrdU).
Base-modifying agents: Chemically alter bases, forming DNA adducts (e.g., EMS, nitrous acid, aflatoxin B1).
Intercalating agents: Insert between bases, distorting DNA structure (e.g., ethidium bromide).

Radiation-Induced DNA Damage

Ultraviolet radiation: Causes pyrimidine dimer formation (covalent bonds between adjacent pyrimidines).
Ionizing radiation: Removes electrons, generating reactive intermediates that damage DNA.

DNA Repair Mechanisms

Overview of DNA Repair Systems

Cells have evolved multiple mechanisms to repair DNA damage, depending on the type and severity of the lesion and the cell cycle stage.

Repair strategies vary for different types of damage.

Light-Dependent Repair

Pyrimidine dimers can be repaired by photoactive repair, which uses the enzyme photolyase and visible light energy.

Photolyase catalyzes the breakage of bonds between thymine dimers.

Base Excision Repair

Base excision repair corrects single damaged bases, such as deaminated bases, using a series of enzymes.

DNA glycosylases recognize and remove damaged bases.
AP endonuclease detects depurination and cleaves the backbone.
DNA polymerase synthesizes the correct base; DNA ligase seals the nick.

Nucleotide Excision Repair (NER)

NER detects distortions in the DNA helix and removes a short segment containing the lesion.

NER endonuclease (excinuclease) cuts the DNA on both sides of the lesion.
Helicase unwinds and removes the damaged segment.
DNA polymerase and ligase fill and seal the gap.

Versatility of NER

NER corrects many types of DNA damage.
Transcription-coupled repair: NER is recruited to regions where transcription is stalled.
Xeroderma pigmentosum: Genetic disorder caused by defective NER, leading to extreme sensitivity to sunlight.

Mismatch Repair

Mismatch repair corrects errors that escape proofreading during DNA replication.

Distinguishes original (methylated) from new (unmethylated) DNA strands.
Incorrect nucleotide in the new strand is excised and replaced.

Process of Mismatch Repair

MutS protein detects the mismatch.
MutH endonuclease introduces a nick in the unmethylated strand.
Exonuclease removes incorrect nucleotides; DNA polymerase and ligase restore the correct sequence.

Double-Strand Break Repair

Overview

Double-strand breaks are severe lesions that split DNA into two fragments. Repair is challenging and can result in loss of nucleotides.

Two main pathways: Nonhomologous end-joining (NHEJ) and Homologous recombination.

Nonhomologous End-Joining (NHEJ)

Proteins (Ku70 and Ku80) bind to broken DNA ends.
Nucleases trim the ends; DNA ligase IV seals the break.
Error-prone: nucleotides may be lost, and incorrect fragments may be joined.

Synthesis-Dependent Strand Annealing (SDSA)

Uses the sister chromatid as a template for repair after DNA synthesis.
Rad51 protein facilitates strand invasion and D loop formation.
Replication and ligation restore the broken chromatid.

Homologous Recombination

Homologous recombination repairs double-strand breaks using a homologous DNA molecule as a template, often resulting in genetic exchange (crossing over).

Strand invasion and D loop formation occur.
DNA synthesis fills in gaps; a Holliday junction is formed and resolved.
Can result in gene conversion or permanent exchange of DNA.

Homologous Recombination and Mobile Genetic Elements

Role in Meiosis

Homologous recombination is essential for chromatin exchange during meiosis, increasing genetic diversity.

Initiation by Double-Stranded Breaks

Recombination begins with asymmetric cleavage by Spo11 protein.
Spo11 recruits proteins to trim single strands; Rad51 stabilizes single-stranded DNA.

Steps of Recombination

Stabilized DNA strands invade homologous DNA, forming a D loop.
Pairing and formation of the Holliday junction.

Strand Extension and Holliday Junction Resolution

DNA synthesis extends the invading strand; branch migration occurs.
Double Holliday junctions may form.
Resolution can result in crossing over (opposite-sense) or no crossing over (same-sense).

Additional info: The study notes above expand on the original slides and text, providing definitions, examples, and context for key terms and processes. All diagrams referenced are described in the text, and the table of trinucleotide repeat disorders is recreated in HTML format for clarity.