DNA Damage and Repair II

DNA Damage and Repair II

Lecture Details
  • Institution: Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University
  • Course: Medical Biochemistry COM 5021
  • Lecture Number: 46
  • Date: 9/30/25
  • Time: 10:10-11:00 am
  • Instructor: Anna Potter, PhD (Email: apotter1@nova.edu, Tampa Bay Regional Campus Office #3614)
Learning Objectives
  • Describe translocations (including Robertsonian translocation) and related diseases.
  • Describe mechanisms of DNA repair, including:
    • Direct repair
    • Base excision repair (BER)
    • Nucleotide excision repair (NER)
    • Mismatch repair
    • Homologous recombination (HR)
    • Non-homologous end joining (NHEJ)
  • Relate human diseases that are due to defects in DNA repair.
Background
  • DNA damage sources:
    • External sources, which can contribute to:
    • Base pair mismatches
    • Single- and double-strand breaks
    • Cross-linking
    • Bulky additions
    • Internal biological processes, including:
    • Cellular metabolism
    • Production of reactive oxygen species (ROS)
    • Replication errors
    • Active topoisomerases
    • Transposons
    • Chromosome translocations
  • Most DNA damage is repaired before mutations become permanent due to efficient DNA repair mechanisms.
Translocations and Diseases
  • Translocation: The rearrangement of genes when a piece of one chromosome breaks off and attaches to another chromosome.
    • The newly formed chromosome is referred to as the translocation chromosome.
    • Types of translocations:
    • Balanced translocation: No genetic material is gained or lost.
    • Unbalanced translocation: There is a gain or loss of genetic material.
  • Chromosome alterations can lead to various diseases.
  • When chromosomes break, the free ends become sticky and may rejoin with other free ends nonspecifically.
  • Major types of chromosomal rearrangements include:
    • Translocation in non-homologous chromosomes.
    • Nonreciprocal translocation: One chromosome takes a piece from another.
    • Reciprocal translocation: Two non-homologous chromosomes trade pieces.
  • Robertsonian translocation involves the attachment of an entire chromosome to another at the centromere (the center of a chromosome where it appears pinched between p and q arms).
    • Example: A translocation between chromosomes 14 and 21 can result in Down syndrome if a baby is born with one normal 14 chromosome and two normal 21 chromosomes along with the translocation chromosome.
Mechanisms of DNA Repair
  • Most types of DNA damage can be repaired. Mechanisms responsible for preserving DNA stability include:
    • DNA polymerase proofreading
    • DNA repair systems
  • Types of DNA damage include:
    • Altered individual bases (alkylated, base analogs)
    • Altered 3-D DNA structure (thymine dimers, intercalating agents)
    • Single-strand breaks
    • Double-strand breaks
  • DNA repair mechanisms:
    • Direct repair
    • Nucleotide excision repair (NER)
    • Base excision repair (BER)
    • Mismatch repair
    • Homologous recombination (HR)
    • Non-homologous end joining (NHEJ)
Direct Repair
  • Direct DNA repair: Reverses alterations without cutting out or replacing nucleotides.
    • Primarily used for repairing thymine dimers.
    • In eukaryotic cells, a light-dependent enzyme called photolyase is used to cut abnormal covalent bonds between the two thymines.
Excision Repair
  • Excision repair: Involves removal of altered bases/nucleotides and replacement with normal DNA.
    • Steps in excision repair include:
    1. Recognition of the lesion by proteins and excision by a nuclease enzyme. Sometimes extra 'good' sequence is also removed.
    2. A DNA polymerase fills in the reconstructing space with correct nucleotides.
    3. DNA ligase seals the final nick (the last phosphodiester bond) between the new and existing strands.
  • Cells have two types of excision repair systems:
    • Base excision repair (BER)
    • Nucleotide excision repair (NER)
Nucleotide Excision Repair (NER)
  • Responsible for repairing DNA damage induced by ultraviolet light (UV).
    • Types/sources of DNA damage include:
    • Bulky DNA lesions formed by UV light or solar radiation (e.g., thymine dimers).
    • Environmental mutagens.
    • Some cancer chemotherapeutic adducts.
  • NER Mechanism for thymine dimers:
    1. NER enzymes identify the thymine dimer.
    2. Enzymes cleave part of the DNA strand containing the dimer.
    3. DNA polymerase adds nucleotides to rebuild the strand.
    4. DNA ligase fills in the gap at the phosphodiester bond.
Base Excision Repair (BER)
  • Base excision repair (BER): Used for correcting minor alterations to individual bases (free radicals, alkylation, base analogs) that are not bulky.
    • Typically occurs when cytosine is deaminated to form uracil.
  • BER Mechanism:
    1. DNA glycosylase enzymes recognize altered bases and cut out the base only, breaking the sugar/base bond.
    2. AP endonuclease recognizes the site missing the base and makes a cut in the sugar/phosphate backbone.
    3. DNA polymerase fills in the correct nucleotide.
    4. DNA ligase repairs the gap in the phosphodiester bond.
Mismatch Repair
  • Mismatch repair: Corrects mismatches that occur right after DNA replication.
    • If a G-T mismatch occurs, the cell determines which one is correct through methylation patterns: the 'old' DNA strand is methylated, while the 'new' unmethylated strand shows mismatches.
  • Mismatch Repair Mechanism:
    1. Mismatch repair enzymes identify mismatched nucleotides and recognize the parental strand.
    2. Enzymes remove part of the strand containing the mismatch.
    3. DNA polymerase rebuilds the new strand with the correct nucleotide.
Homologous Recombination Repair
  • Homologous recombination repair: Fixes double-stranded breaks using homologous chromosomes as templates.
    • RecBCD enzymes recognize double-stranded breaks, partially degrade one strand on each side, and create single-stranded overhangs.
    • RecA binds to the single-stranded end and promotes invasion of the homologous chromosome.
    • RuvABC, DNA polymerase, and ligase help recreate the gaps and resolve the structure, yielding a repaired chromosome containing a piece of homologous DNA.
    • This process is similar to crossing over during meiosis.
Non-Homologous End-Joining Repair
  • Non-Homologous end-joining (NHEJ): Used to fix double-stranded breaks without the need for a sister chromatid.
    • Broken ends are simply glued back together, stabilized by end-binding proteins and cross-bridging proteins.
    • Ends are processed, filled, and ligated.
    • Advantages and Disadvantages:
    • Advantage: Can occur at any time in the cell cycle.
    • Disadvantage: May lead to small deletions near the break site.
Human Diseases Due to Defects in DNA Repair
  • Xeroderma Pigmentosum (XP): Caused by defects in NER; characterized by severe skin abnormalities upon sun exposure, resulting from inability to repair UV-induced DNA damage. The condition can lead to mutations, causing cells to die or become cancerous. Mutations found in 7 different genes within the NER pathway are associated with XP. NER in eukaryotic cells involves as many as 20 different proteins.
  • Severe Combined Immunodeficiency (SCID): Associated with dysfunctional NHEJ, leading to absent or low T cells and B cells. Possible treatment includes bone marrow transplant.
  • Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC): Also known as Lynch Syndrome, caused by dysfunctional nucleotide mismatch repair. Individuals at an increased lifetime risk for colorectal, endometrial, stomach, ovarian, urinary tract, and other cancers.
  • BRCA1/BRCA2 mutations: Associated with dysfunctional homologous recombination, leading to increased susceptibility to breast, ovarian, and other cancers. BRCA proteins coordinate with Rad51 during strand invasion, facilitating repair and maintaining controlled cell growth and division.