DNA Repair, Recombination, and Rearrangement

Introduction

This chapter explores the molecular mechanisms by which cells maintain the integrity of their genetic material. DNA is constantly exposed to damaging agents, and its repair is essential for cell survival and prevention of diseases such as cancer. Additionally, recombination and rearrangement processes contribute to genetic diversity and adaptation.

DNA Repair

DNA Damage and Strategies for Repair

DNA damage can arise from various sources, and cells have evolved multiple strategies to repair such damage and preserve genetic information.

• Sources of DNA Damage:

  • Radiation: Ultraviolet (UV) and ionizing radiation can cause breaks and chemical modifications in DNA.

  • Environmental Chemicals: Agents such as alkylating compounds and carcinogens can modify DNA bases.

  • Endogenous Reactive Oxygen Species (ROS): Byproducts of cellular metabolism can oxidize DNA bases.

• Importance of DNA Repair:

  • DNA serves as the cell's information storage molecule; repair mechanisms are essential to maintain genetic fidelity.

Major DNA Repair Processes

Cells utilize several distinct repair pathways to address different types of DNA damage:

• Photoreactivation: Direct reversal of UV-induced damage using light-dependent enzymes.

• Removal of Alkylguanines: Specific enzymes remove alkyl groups from guanine bases.

• Nucleotide Excision Repair (NER): Removes bulky lesions such as thymine dimers and chemical adducts.

• Base Excision Repair (BER): Excises and replaces damaged or incorrect bases.

• Mismatch Repair: Corrects errors introduced during DNA replication.

• Double-Strand Break Repair: Repairs breaks in both DNA strands, often via homologous recombination.

• Daughter Strand Gap Repair: Uses undamaged template DNA to fill in gaps left during replication.

Types of DNA Damage and Repair Processes

DNA can be damaged in various ways, each requiring specific repair mechanisms. The following table summarizes common types of damage and their corresponding repair processes:

Endogenous DNA-Damaging Reactions

Endogenous reactions within cells can damage DNA, often as a result of normal metabolic processes.

• Hydrolysis: Leads to deamination and depurination of bases.

• Oxidation: ROS can oxidize guanine to 8-oxoguanine, which mispairs with adenine.

• Alkylation: Addition of alkyl groups to bases, such as O6-methylguanine.

Structures of Thymine Dimer Photoproducts

UV radiation can induce the formation of thymine dimers, which distort the DNA helix and block replication.

• Cyclobutane Thymine Dimer: Two adjacent thymine bases become covalently linked, causing a significant distortion.

• 6-4 Photoproduct: Another type of dimer that is highly mutagenic and can lead to inaccurate replication.

Example: Thymine dimers are a major cause of skin cancer due to UV exposure.

Direct Repair of DNA Damage by Alkyltransferases

Alkylating agents can modify DNA bases, leading to mutagenesis or cell death if not repaired.

• Alkyltransferases: Enzymes that remove alkyl groups from guanine, restoring normal base pairing.

• Example: O6-methylguanine is repaired by O6-methylguanine-DNA methyltransferase.

Nucleotide Excision Repair (NER)

NER is a versatile repair system that removes bulky DNA lesions, such as thymine dimers and chemical adducts.

• Process:

  1. Recognition of DNA distortion.

  2. Excision of a short single-stranded DNA segment containing the lesion.

  3. DNA synthesis to fill the gap.

  4. DNA ligation to restore continuity.

• Example: Repair of UV-induced thymine dimers in E. coli by the UvrABC excinuclease complex.

Base Excision Repair (BER) of Oxidative Damage

BER corrects small, non-helix-distorting base lesions, such as those caused by oxidation.

• 8-oxoguanine (oxoG): A common oxidative lesion that can mispair with adenine, leading to mutations.

• OGG1 DNA Glycosylase: Recognizes and excises oxoG bases in humans.

Methyl-Directed Mismatch Repair in Escherichia coli

Mismatch repair corrects errors that escape proofreading during DNA replication.

• Mechanism:

  1. MutHLS complex identifies the newly synthesized strand by its lack of methylation at GATC sites.

  2. Excision of the DNA segment containing the mismatch.

  3. DNA polymerase III and ligase fill and seal the gap.

Double-Strand Break (DSB) Repair Through Homologous Recombination (HR)

DSBs are repaired using a homologous DNA template, ensuring accurate restoration of genetic information.

• Homologous Recombination: Uses a sister chromatid or homologous chromosome as a template.

• Clinical Relevance: Defects in BRCA1 and BRCA2 genes increase risk for breast and ovarian cancer.

Daughter-Strand Gap Repair

This process allows replication to continue past damaged sites by using the undamaged template from the sister chromatid.

• RecA Protein: Facilitates strand exchange and repair.

Recombination

Site-Specific Recombination

Site-specific recombination involves the exchange of DNA at particular sequences, often mediated by specialized enzymes.

• Example: Lysogeny in bacteriophage λ, where integrase inserts the viral genome into the host chromosome at the attB site.

Holliday Model for Homologous Recombination

The Holliday model describes the molecular steps of homologous recombination between paired chromosomes.

1. DNA strands are nicked at the same site on both chromosomes.

2. Strand invasion and partial unwinding occur.

3. Enzymatic ligation forms a crossed-strand intermediate (Holliday junction).

4. The junction can migrate along the duplex, and isomerization leads to recombinant or nonrecombinant heteroduplexes.

Visualization: Holliday junctions can be observed by electron microscopy.

Gene Rearrangements

Generation of Antibody Diversity Through Gene Rearrangements

Antibody diversity is achieved by recombination of variable (V) and joining (J) gene segments during B cell differentiation.

• Process:

  1. One of ~300 V sequences and one of four J sequences are combined.

  2. Intervening DNA is spliced out and removed from all progeny of that cell line.

  3. Upstream V and downstream J sequences are retained but not transcribed.

Joining to Achieve Additional Diversity

Proteins RAG1 and RAG2 catalyze double-strand breaks, initiating recombination of V and J segments. Variability in joining further increases diversity.

Transposable Genetic Elements and Chromosome Rearrangements

Transposons are DNA sequences that can move within the genome, promoting rearrangements.

• Homologous Recombination: Can occur between two copies of the same transposable element, leading to insertions or deletions.

• Transposase: Enzyme that mediates transposon movement and generates direct repeats at insertion sites.

• Complex Transposons: Encode additional proteins, such as antibiotic resistance.

Retroviruses and Long Terminal Repeats (LTRs)

Retroviruses integrate into host chromosomes using LTRs, which facilitate insertion and amplification.

Gene Amplification via Unequal Sister-Chromatid Exchange

Unequal crossing over during recombination can lead to gene amplification, increasing gene copy number.

Tools of Biochemistry

Targeted Insertion of Genes into a Genome

Genetic engineering techniques allow for the targeted insertion or knockout of genes, such as replacing the hprt gene with the neor gene.

CRISPR-Cas9 System

CRISPR technology utilizes the Cas9 enzyme to catalyze site-specific double-stranded DNA cleavage. This system is widely used for gene knockout, site-directed mutagenesis, and gene insertion.

• Example: The CRISPR-Cas9 system from Streptococcus thermophilus is a model for genome editing.

Key Equations and Concepts

• Base Pairing: ,

• DNA Repair Rate: Additional info: This equation is a general representation of enzyme-catalyzed repair kinetics.

Summary Table: DNA Repair Mechanisms

Additional info: This summary table is inferred from the chapter's content and standard biochemistry knowledge.