dna repair 2

DNA Damage and Repair II

Course Information

  • Institution: Kiran C. Patel College of Osteopathic Medicine
  • Course: Medical Biochemistry COM 5021
  • Lecture Number: 46
  • Date and Time: 9/30/25, 10:10-11:00 am
  • Instructor: Anna Potter, PhD, Assistant Professor
  • Email: apotter1@nova.edu
  • Campus: Tampa Bay Regional Campus
  • Office Number: 3614

Learning Objectives

  1. Describe translocations (including Robertsonian translocation) and associated diseases.
  2. 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)
  3. Relate human diseases resulting from defects in DNA repair mechanisms.

Background on DNA Damage

  • Sources of DNA Damage:
    • External sources can lead to various types of DNA damage, resulting in:
      • Base pair mismatches
      • Single- and double-strand breaks
      • Cross-linking
      • Bulky additions.
    • Biological Processes Contributing to DNA Damage:
      • Cellular metabolism
      • Production of reactive oxygen species (ROS)
      • Replication errors
      • Active topoisomerases
      • Transposons
      • Chromosome translocations.
  • Most DNA damage is effectively repaired before it results in permanent mutations due to active DNA repair mechanisms.

Translocations and Associated Diseases

  • Definition: Translocations involve the rearrangement of genes when a segment of one chromosome breaks off and attaches to another chromosome.
  • The rearranged chromosome is referred to as a translocation chromosome.
  • Types of Translocations:
    • Balanced Translocation: No genetic material is gained or lost during the rearrangement.
    • Unbalanced Translocation: There is a gain or loss of genetic material resulting from the translocation.
Chromosome Alterations
  • Chromosome breakage can result in free ends that are "sticky" and rejoin with other broken ends nonspecifically.
Types of Gene Translocations
  • Translocations can occur in non-homologous chromosomes.
    • Nonreciprocal Translocation: One chromosome takes a piece from another chromosome without reciprocation.
    • Reciprocal Translocation: Two non-homologous chromosomes exchange random pieces.
Robertsonian Translocation
  • In a Robertsonian translocation, an entire chromosome attaches to another at the centromere, which is the constricted region of a chromosome that divides it into p and q arms.
  • Example of Robertsonian Translocation:
    • Involves chromosomes 14 and 21.
    • A child born with one normal chromosome 14 and two normal chromosome 21, alongside a translocation chromosome, may develop Down syndrome.

Mechanisms of DNA Repair

  • Most types of DNA damage can be repaired.
  • Mechanisms Responsible for DNA Stability:
    • DNA polymerase proofreading
    • Various DNA repair mechanisms undertake the repair of numerous damage types, which may include:
      • Altered individual bases (e.g., alkylated bases, base analogs)
      • Altered three-dimensional structures of DNA (e.g., thymine dimers, intercalating agents)
      • Single-strand breaks
      • Double-strand breaks
  • Categories of 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
  • Definition: Direct DNA repair involves reversing the alteration without excising or replacing nucleotides.
  • Primarily used for the repair of thymine dimers.
  • Eukaryotic cells utilize a light-dependent enzyme called photolyase to break the abnormal covalent bonds formed between adjacent thymines.
Excision Repair
  • Definition: Excision repair refers to the removal of an altered base or nucleotide, followed by replacement with the correct DNA sequence.
  • Mechanism Steps:
    1. Recognition of the lesion by one or more proteins and subsequent excision by a nuclease enzyme.
      • Sometimes, extra "good" sequence is removed during this step.
    2. A DNA polymerase fills in the gap with the correct nucleotides.
    3. DNA ligase seals the final nick (the last phosphodiester bond) between the newly added and existing strands.
  • Types of Excision Repair Systems:
    1. Base excision repair (BER)
    2. Nucleotide excision repair (NER)
Nucleotide Excision Repair (NER)
  • Function: NER repairs DNA damage induced by ultraviolet (UV) light.
  • Damage Sources:
    • Forms bulky lesions like thymine dimers caused by UV light or solar radiation
    • Environmental mutagens
    • Chemotherapeutic drugs that create adducts with DNA.
  • Deficiency in NER:
    • Associated with xeroderma pigmentosum, a genetic disorder that predisposes individuals to skin cancer upon UV exposure.
  • NER Mechanism for Thymine Dimers:
    1. NER enzymes identify the thymine dimer.
    2. Cleavage of the strand containing the dimer occurs by the NER enzymes.
    3. DNA polymerase adds nucleotides to reconstruct the strand.
    4. DNA ligase fills in the gap at the phosphodiester bond.
Base Excision Repair (BER)
  • Definition: BER is utilized to correct minor alterations to individual bases that are not bulky, such as those caused by free radicals or alkylation.
  • Common Scenario: The deamination of cytosine forming uracil.
  • BER Mechanism Steps:
    1. DNA glycosylase enzymes recognize the altered bases, with different glycosylases for different altered bases.
    2. The glycosylase cuts out the mispaired base by breaking the sugar/base bond.
    3. AP (apurinic/apyrimidinic) endonuclease recognizes the missing base site and makes a cut in the sugar/phosphate backbone.
    4. DNA polymerase fills in the correct nucleotide.
    5. DNA ligase repairs the gap in the phosphodiester bond.
Mismatch Repair (MMR)
  • Function: Mismatch repair corrects mismatches that occur soon after DNA replication.
  • Distinguishing Strands: The mechanism can distinguish between the newly synthesized (unmethylated) and parental (methylated) strands.
    • In the presence of a G-T mismatch, the mismatch repair system identifies which nucleotide is incorrect based on methylation patterns.
  • MMR Mechanism Steps:
    1. MMR enzymes identify mismatched nucleotides and the parental strand.
    2. The MMR enzymes remove part of the strand containing the mismatch.
    3. DNA polymerase rebuilds the strand with the correct nucleotide.
Homologous Recombination Repair (HRR)
  • Purpose: HRR repairs double-stranded breaks using homologous chromosomes as repair templates.
  • A specific enzyme complex, RecBCD, recognizes double-stranded breaks and partially degrades one strand on each side to create single-stranded overhangs.
  • RecA binds to the single-stranded end and promotes invasion of the homologous chromosome.
  • RuvABC, along with DNA polymerase and ligase, helps to reconstruct the gaps and resolve the structure.
Non-Homologous End-Joining Repair (NHEJ)
  • Functionality: NHEJ repairs double-stranded breaks by simply gluing the broken ends back together without requiring a sister chromatid.
  • Process Includes:
    • Binding of end-binding proteins to each side of the break for stabilization.
    • Recruitment of cross-bridging proteins to prevent drift of the two ends.
    • Processing, filling, and ligation of the ends.
  • Advantages: NHEJ can occur at any point in the cell cycle.
  • Disadvantages: This process can lead to small deletions near the break site.

Human Diseases Associated with DNA Repair Defects

  • Xeroderma Pigmentosum (XP):

    • Disorder related to defects in nucleotide excision repair (NER).
    • XP patients possess one of several rare mutations in components of the NER pathway, which results in severe skin abnormalities upon UV exposure, causing freckling, ulceration, and skin cancer due to the lack of repair.
    • Research has identified mutations in any of seven different genes that can lead to XP.
    • The NER mechanism in eukaryotes is complex, involving as many as 20 different proteins.

  • Severe Combined Immunodeficiency (SCID):

    • Caused by dysfunctional non-homologous end joining (NHEJ), leading to the absence or low levels of T and B cells.
    • Possible treatment option includes bone marrow transplant.

  • Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC):

    • Also known as Lynch Syndrome, caused by dysfunctional nucleotide mismatch repair.
    • Individuals with this condition exhibit an increased lifetime risk for several cancers, including colorectal, endometrial, stomach, ovarian, urinary tract cancers, and more.

  • BRCA1/BRCA2 Mutations:

    • Dysfunctional homologous recombination is frequently linked to these mutations, primarily associated with various cancers.
    • BRCA proteins work synergistically with Rad51 during strand invasion to aid in repair, thereby preventing uncontrolled cell growth and division in breast, ovarian, and other types of cells.