Lecture 3 (2) PDF: Molecular Mechanisms of DNA and Chromosome Damage and Repair

Molecular Mechanisms of DNA and Chromosome Damage and Repair 1 GENERAL OVERVIEW OF DNA STRAND BREAKS DNA as the Principal Target for Radiation Effects 1. DNA is the primary target for the biological effects of radiation, including cell death, carcinogenesis, and mutation. 2. Understanding radiat...

Molecular Mechanisms of DNA and Chromosome Damage and Repair 1 GENERAL OVERVIEW OF DNA STRAND BREAKS DNA as the Principal Target for Radiation Effects 1. DNA is the primary target for the biological effects of radiation, including cell death, carcinogenesis, and mutation. 2. Understanding radiation's effects on DNA begins with describing the breaks in DNA caused by charged particle tracks and chemical species produced by radiation. Radiation It's can critcial breah DNA's structure becaus DNA controls 2 , cell functions . A > - G-C GENERAL OVERVIEW OF DNA STRAND phosphate , sugar BREAKS . Structure of DNA • DNA is the most important material making up the chromosome and serve as the master blue print of the cell. • It consists of two strands held together by hydrogen bonds between bases. • The "backbone" of each strand consists of alternating sugar and phosphate groups. • Four bases, adenine, thymine, cytosine, and guanine, determine the genetic code. • Bases on opposite strands must be complementary, with adenine pairing with thymine and guanine pairing with cytosine. • In Radiation Biology, DNA is the critical target for radiation. 3 GENERAL OVERVIEW OF DNA STRAND BREAKS Types of DNA Lesions Induced by Radiation • Radiation induces various types of lesions in DNA. • Most lesions are repaired successfully by cells. • The number and types of DNA lesions detected immediately after a 1 Gy dose of x-rays are listed. 1. Single-strand breaks (SSBs) are relatively common but are often repaired using the opposite strand as a template. 2. Double-strand breaks (DSBs) are believed to be the most critical lesions, and their interaction may result in cell death, carcinogenesis, or mutation. • DSBs are induced linearly with dose and are formed by single tracks of ionizing radiation. 4 MEASURING DNA STRAND BREAKS DNA Strand Break Measurement Techniques: • Various techniques have been used to measure DNA strand breaks, including 1. sucrose gradient sedimentation, 2. alkaline and neutral filter elution, 3. nucleoid sedimentation, 4. pulsed-field gel electrophoresis (PFGE) 5. single-cell gel electrophoresis (comet assay) 6. DNA Damage-Induced Nuclear Foci * (PFGE, single-cell gel electrophoresis and DNA DamageInduced Nuclear Foci are currently used to measure DNA strand breaks) 5 MEASURING DNA STRAND BREAKS Pulsed-Field Gel Electrophoresis (PFGE) : • PFGE is widely used to detect and study DNA double-strand breaks (DSBs). • It involves electrophoretic elution of DNA from agarose plugs containing irradiated cells. • PFGE separates DNA fragments by size, assuming that DSBs are induced randomly. • The fraction of DNA released from the agarose plug is directly proportional to the radiation dose. • DNA DSB rejoining kinetics exhibit fast initial rates followed by slower phases. • Chromosomal damage, such as translocations and exchanges, is associated with slowly rejoining DSBs. 6 MEASURING DNA STRAND BREAKS Single-cell electrophoresis (comet assay) : • The comet assay is used to detect differences in DNA damage and repair at the single-cell level. • It is advantageous for studying biopsy specimens from tumors. • Cells are exposed to ionizing radiation, embedded in agarose, and lysed to quantify DNA damage and repair. • The extent of DNA damage is directly proportional to the migration of DNA in the agarose, forming a cometlike appearance. • The comet assay is highly sensitive to single-strand breaks (SSBs) and alkaline-sensitive sites and can measure DNA DSB repair when lysis conditions change from alkaline to neutral pH 7 MEASURING DNA STRAND BREAKS DNA Damage-Induced Nuclear Foci: • Radiation-induced nuclear foci represent complexes of signaling and repair proteins that localize to sites of DNA strand breaks in the nucleus. • Assaying for foci formation has advantages, including ease of protocol and applicability to tissue sections and individual cells. • Common proteins assayed for foci formation include γH2AX and 53BP1. • The number of foci correlates with the presence of DNA doublestrand breaks (DSBs) and reflects DSB repair kinetics. • Foci formation can be quantified by fluorescence microscopy and flow cytometry. • The γH2AX or 53BP1 foci that form in a damaged cell correlate with the presence of DSBs 8 DNA REPAIR PATHWAYS • Mammalian cells have specialized pathways for sensing, responding to, and repairing various types of DNA damage, including base damage, single-strand breaks (SSBs), doublestrand breaks (DSBs), sugar damage, and DNA–DNA crosslinks. • Different repair pathways are used to repair DNA damage, depending on the stage of the cell cycle: 1. Base Excision Repair 2. Nucleotide Excision Repair 3. DNA Double-Strand Break Repair A. Nonhomologous End-Joining B. Homologous Recombination Repair 9 Base Excision Repair: • BER is a DNA repair pathway that addresses base damage. • Complementary base pairing is essential: A with T and G with C. • The repair process involves recognizing and removing damaged bases. • Single-Base Repair : • A putative single-base mutation (U) is first removed by a glycosylase/DNA lyase. • Subsequent steps include sugar residue removal (APE1), correct nucleotide insertion (DNA polymerase β), and ligation (DNA ligase III–XRCC1). • • • • Multiple-Base Repair : In cases of multiple nucleotide mutations (e.g., UU), repair is more complex. Repair synthesis involves RFC/PCNA/DNA polymerase δ/ε. Overhanging flap structure removal (FEN1) and strand sealing (ligase I) complete the process. Efficient Repair of Ionizing Radiation-Induced Base Damage: BER efficiently repairs base damage caused by ionizing radiation. Defects in BER can increase mutation rates but usually do not cause cellular radiosensitivity. Exception: Mutation in XRCC1 gene, which may lead to a 1.7-fold increase in radiation sensitivity. • XRCC1 may also play a role in repairing single-strand breaks (SSBs) and other repair processes • • • • 1 0 DNA REPAIR PATHWAYS ( Nucleotide Excision Repair (NER) A DNA repair pathway that removes bulky adducts from DNA, such as pyrimidine dimers formed by UV light. Two types of NER: 1. Global genome repair (GGR): Removes lesions from all DNA, regardless of transcription status. 2. Transcription-coupled repair (TCR): Removes lesions from actively transcribed DNA. Mechanism of NER 1. 2. 3. 4. 5. Damage recognition: A complex of DNA repair proteins recognizes the lesion. Incisions: Endonucleases cleave the DNA strand on either side of the lesion, creating a 24-32 nucleotide gap. Removal of the lesion: The helicase XPD unwinds the DNA and the lesion is removed. Repair synthesis: DNA polymerase fills in the gap with new nucleotides. DNA ligation: DNA ligase seals the nick in the DNA backbone. Consequences of Defective NER • • • • Increased sensitivity to UV light, alkylating agents, and other carcinogens. Increased risk of skin cancer and other malignancies. Human DNA repair deficiency disorders such as xeroderma pigmentosum. Patients with xeroderma pigmentosum are hypersensitive to UV light and must take precautions to avoid sun exposure. 1 1 DNA REPAIR PATHWAYS (DNA Double-Strand Break Repair) In eukaryotic cells, the two predominant pathways for the repair of DNA DSBs are: 1- Homologous recombination repair (HRR) 2- Nonhomologous end-joining (NHEJ) Nonhomologous end-joining (NHEJ) • Repairs DNA DSBs by joining the ends of the broken DNA. • Error-prone process. • Predominant in G1 phase of cell cycle in mammalian cells. Homologous recombination repair (HRR) • Requires an undamaged DNA strand as a template for repair. • Error-free process. • Predominant in lower eukaryotes and late S/G2 phase of cell cycle in mammalian cells. 1 2 DNA REPAIR PATHWAYS (DNA Double-Strand Break Repair) 1- Nonhomologous end-joining (NHEJ) A DNA repair pathway that joins DNA ends together without requiring homology. DNA Damage Response and Repair o DNA damage sensors: ATM and ATR are protein kinases that are activated by DNA DSBs and promote DNA repair and cell cycle arrest. o 53BP1: A protein that promotes NHEJ and inhibits HRR. NHEJ Steps 1. End recognition: The Ku heterodimer binds to the DNA ends. 2. Recruitment of DNA-PKcs: DNA-PKcs is recruited to the DNA ends by Ku. 3. End processing: Artemis, an endonuclease, trims the DNA ends. 4. Fill-in synthesis: Gaps in the DNA are filled in by DNA polymerase µ or λ. 5. Ligation: The DNA ends are ligated together by DNA ligase IV, XRCC4, XLF, and PNK. NHEJ Characteristics o NHEJ is error-prone. o NHEJ plays an important role in generating antibody diversity. o NHEJ is primarily found in the G1 phase of the cell cycle. 1 3 (DNA Double-Strand Break Repair) 2- Homologous Recombination Repair (HRR) A DNA repair pathway that uses a homologous sister chromatid as a template to repair DSBs. HRR Steps 1. End resection: Nucleases resect the DNA ends to create 3' single-strand DNA overhangs. 2. Rad51 loading: BRCA1 and BRCA2 facilitate the loading of Rad51 onto the 3' single-strand DNA overhangs. 3. Strand invasion: Rad51 forms a nucleofilament and invades the homologous DNA strand on the sister chromatid. 4. DNA synthesis: DNA polymerases extend the invading strands, using the homologous strand as a template. 5. Holliday junction resolution: The Holliday junctions are resolved by MMS4 and MUS81, resulting in non-crossover or crossover events. 6. Gap filling and ligation: Gaps in the DNA are filled in by DNA polymerases and the DNA ends are ligated together by DNA ligases. HRR Characteristics • • • Requires an undamaged DNA strand as a template for repair. Error-free process. Predominant in lower eukaryotes and late S/G2 phase of cell cycle in mammalian cells. 1 4 DNA REPAIR PATHWAYS (Crosslink Repair) Crosslink Repair DNA crosslinks are covalent bonds between two nucleotides in DNA • Crosslinks can be either intrastrand (within the same strand of DNA) or interstrand (between two strands of DNA). • Crosslinks can be caused by a variety of factors, including chemicals, UV light, and ionizing radiation Consequences of DNA crosslinks • • DNA crosslinks can interfere with replication and transcription. Unrepaired crosslinks can lead to cell death. Intrastrand crosslinks Intrastrand crosslinks can be repaired by DNA polymerase switching to a template without the crosslink or by translesion synthesis. Interstrand crosslinks Interstrand crosslinks are repaired by NER in G1 phase. 1 5 DNA REPAIR PATHWAYS (Mismatch Repair ) Mismatch Repair (MMR) is a DNA repair pathway that removes base-base and small insertion mismatches that occur during replication and homologous recombination. MMR Steps 1. Mismatch recognition: Sensors, such as MutS and MSH2-MSH6, identify the mismatch. 2. Recruitment of MMR factors: MMR proteins, such as MLH1-MLH3 and EXO1, are recruited to the mismatch site. 3. Excision of the mismatch: EXO1 excises the newly synthesized strand harboring the mismatch. 4. Resynthesis and ligation: DNA polymerase δ/ε and PCNA fill in the gap and DNA ligase ligates the DNA ends together. Consequences of MMR defects • Microsatellite instability (MSI) (A condition characterized by instability in the length of microsatellites, which are short repeating sequences of DNA) 1 6 • Cancer, especially hereditary nonpolyposis colon cancer (HNPCC) Relationship between DNA Damage and Chromosome Aberrations • • • • Cell killing correlates better with DSBs than SSBs. Agents that produce SSBs efficiently but few DSBs kill very few cells. Cells defective in DNA DSB repair are more sensitive to killing by ionizing radiation and have increased numbers of chromosome aberrations. DSBs can lead to chromosomal aberrations that cause problems at cell division. 1 7 Chromosomes and Cell Division • • • • • DNA helix is made up of molecules of sugar and phosphates that hold the bases that carry the genetic code. Chromosomes are condensed forms of DNA that are visible during cell division. Chromosomes are not free to move about within the nucleus during interphase but are restricted to "domains." Mitosis is the process of cell division that results in two identical daughter cells. Mitosis is divided into four phases: prophase, metaphase, anaphase, and telophase. 1. Prophase • Chromatin condenses into light coils. • Each chromosome has a centromere and two arms. • Nuclear membrane and nucleoli disappear. 2. Metaphase • Chromosomes move to the center of the cell (equator). • Spindle forms and connects the poles of the cell. • Chromosomes are stabilized at the equator. • Centromeres divide. 3. Anaphase • Chromosomes are pulled to the poles of the cell by spindle fibers. • Arms of the chromosomes trail behind. 4. Telophase 1 8 • Chromosomes uncoil at the poles of the cell. • Nuclear membrane and nucleoli reappear. • Chromosome coils unwind. THE ROLE OF TELOMERES Telomeres • Telomeres are caps at the ends of chromosomes that protect them from damage and degradation. • Telomeres are made up of repeating sequences of DNA. • Telomeres shorten each time a cell divides. • When telomeres become too short, the cell can no longer divide and dies. Telomeres and Aging • Telomere shortening is associated with aging. • Stem cells and cancer cells have an enzyme called telomerase that can lengthen telomeres. • Telomerase activity is low or absent in normal somatic cells. Telomeres and Cancer • Almost all cancer cells have telomerase activity. • Telomerase activity allows cancer cells to divide indefinitely. 1 9 RADIATION-INDUCED CHROMOSOME ABERRATIONS Radiation-Induced Chromosome Aberrations • Chromosome aberrations can be seen at the first metaphase after exposure to radiation. • Mammalian cells have many chromosomes, making it difficult to study radiation damage. • Plant cells have fewer and larger chromosomes, making them more suitable for studying radiation damage. Types of Aberrations • Chromosome aberrations occur if a cell is irradiated early in interphase, before the chromosome material has been duplicated. • Chromatid aberrations occur if a cell is irradiated later in interphase, after the chromosome material has doubled. Steps in chromosome aberration formation: 1. Ionizing radiation produces DNA double-strand breaks (DSBs) in the chromosomes. 2. The broken ends of the chromosomes are sticky and can rejoin with any other sticky end. 3. If the broken ends rejoin in their original configuration, no aberration is visible. 4. If the broken ends fail to rejoin, a deletion aberration is produced. 5. If the broken ends rejoin with other broken ends, complex chromosome aberrations are produced. 2 0 EXAMPLES OF RADIATION-INDUCED ABERRATIONS Lethal aberrations: 1. Dicentric: An aberration in which two chromosomes are fused together at their centromeres. 2. Ring: An aberration in which a chromosome is formed into a ring shape. 3. Anaphase bridge: An aberration in which a chromatid is stretched between the two poles of the cell at anaphase. Nonlethal aberrations: 1. Symmetric translocation: An aberration in which two chromosomes exchange material. 2. Small interstitial deletion: An aberration in which a small portion of a chromosome is lost. 2 1