DNA Damage and Repair Mechanisms PDF

DNA Damage and Repair Mechanisms Bindong Liu, PhD Professor, Microbiology and Immunology Email: [email protected]; Tel: 615-327-6877 June 2024  Learning Objectives ❑ Describe endogenous and exogenous sources of DNA damage and the...

DNA Damage and Repair Mechanisms Bindong Liu, PhD Professor, Microbiology and Immunology Email: [email protected]; Tel: 615-327-6877 June 2024  Learning Objectives ❑ Describe endogenous and exogenous sources of DNA damage and the types of lesions that they cause ❑ Explain the consequences of different forms of DNA damage ❑ Outline the basic steps of different DNA repair systems ❑ Identify diseases associated with DNA repair defects Maintaining genetic stability is critical for life Genetic stability is necessary for life o Extremely accurate mechanism for replication of DNA o Highly efficient DNA repair Tens of thousands of mutations are repaired every day Less than 0.02% accumulate as a permanent mutation in the human DNA genome Defects in DNA repair links many human diseases e.g., BRCA1 and BRCA2 mutations cause homologous recombination defects leading to breast and ovarian cancer  Source of DNA damage/mutations ▪ Endogenous/spontaneous - Mutant cells that arise in nature (e.g., polymerase mistakes) ▪ Exogenous/induced - Mutant cells that arise as a result of exposure to a mutagen (an agent that causes mutations) ❖ Caused by physical or chemical mutagens Endogenous/spontaneous DNA damage Red arrow: oxidative damage Green arrow: methylation Blue arrow: hydrolytic attack The width of arrow= frequency Molecular Biology of the Cell (© Garland Science 2015) Although highly stable, DNA is still susceptible to spontaneous changes Depurination: loses purine bases (adenine and guanine) Deamination: the removal of an amino group from nucleotide  Depurination and Deamination ▪ If not repaired Deaminated C: U will pair with A and cause G to A mutation. Depurinated A: DNA replication “skip” to cause deletion Molecular Biology of the Cell (© Garland Science 2015)  Exogenous/induced DNA damage ▪ Chemical or physical agents that induce mutations ▪Alter the primary sequence of DNA ▪Promotes errors in replication or repair of DNA  Three classes of Chemical Mutagens ▪ Nucleotide-base analogs 5-bromouracil (5-BrU) leads to mispairing and DNA replication mistakes ▪ Frameshift mutagens Ethidium bromide (EB) insert between the bases causes addition or deletion ▪ DNA-reactive chemicals Nitrous acid (HNO2) changes the chemical structure of the base pair abnormally  Chemical Mutagens: Nucleotide-base analogs ▪ 5-bromouracil (5-BrU) structure similar to thymine ▪ 5-BrU replaces thymine (T) ▪ 5-BrU pair with guanine (G) or adenine (A) (https://www.biologyonline.com) ▪ Cause A:T to G:C mutation  Chemical Mutagens: Frameshift/Intercalating mutagens ▪ Proflavin, acridine, ethidium bromide (EB) flat molecules ▪ Insert into DNA duplex ▪ Cause “stretching” of DNA duplex ▪ Insertion or deletion  Chemical Mutagens: DNA-reactive chemicals ▪ Nitrous Acid (HNO2) cause deamination ▪ Convert: Cytosine to Uracil Adenine to Hypoxanthine ▪ Cause mutation (https://www.mun.ca/biology/)  Physical Mutagens: ▪ Radiation was the first mutagens known, reported 1920’s ▪ Two types of radiations Electromagnetic radiations: UV radiation Ionizing radiations: X- and gamma-rays Physical Mutagens: Electromagnetic radiations: ▪ Nonionizing ▪ UV radiation reacts with DNA and other biological molecules ▪ UV radiation forms TT dimer or CC dimer  Physical Mutagens: Electromagnetic radiations: UV radiation cause pyrimidine dimers ▪ Most TT or CC dimers will be The dimers initiate nucleotide excision repair corrected ▪ TT dimer gets read correctly ▪ CC often misread as TT If the dimers is not fixed: ❖ TT will be read normally (no mutations) ▪ CC to TT shows up in p53 tumor suppressor gene skin cancers. ❖ CC misread as TT (Copyright © 2022 The Bumbling Biochemist)  Physical Mutagens: Ionizing radiations: ▪ X- and gamma-rays ▪ Produce reactive ions (charged atoms or molecules) ▪ Damage: base and sugar single strand break double-strand break ▪ Severity depends upon the dose received Signal Transduction and Targeted Therapy volume 5, 60 (2020)  DNA Repair It is the interplay between various substrates, enzymes and co-factors etc., to correct/repair the errors. Includes several different processes DNA repair has a remarkable efficiency Diminished capacity for DNA repair in humans has been linked to many human diseases DNA double helix is the foundation for DNA repair o Each strand stores a separate copy of the information. Lesions (mutations) can, therefore, be cut out, and a correct strand resynthesized from information on the undamaged strand o All cells use dsDNA except a few small viruses  Types of DNA Repair Strand-directed mismatch repair (MMR) Base excision repair (BER) Nucleotide excision repair (NER) Non-homologous end joining (NHEJ) Homologous recombination (HR)  Strand-Directed Mismatch Repair Corrects mismatched bases Also called DNA Mismatch Repair Single base or 2-5 bases small loops CGGUTTACGGTAA | || || || | |||| Steps: GCCGAATGCCATT Scan newly made strand Endonuclease cuts backbone Exonuclease removes bases to mutation Fill in the gap with usual replication enzymes like ligase, polymerase,  Strand-Directed Mismatch Repair MutS binds specifically to a mismatched base pair MutL scans the nearby DNA for a nick MutL triggers the degradation of the nicked strand Nicked strand is degraded all the way back through the mismatch, and replication errors are selectively removed Fill in the gap with usual replication enzymes like Ligase, polymerase Molecular Biology of the Cell (© Garland Science 2015) Human disease related to DNA-Mismatch-Repair Hereditary non-polyposis colon cancer (HNPCC) an autosomal dominant disease the most common caused by mutations in hMLH1 (MutL) or hMSH2 (MutS)  Excision Repair Mechanisms Damage is excised, and the original DNA sequence is restored using the undamaged strand as its template. Excision repair mechanisms are very common Two kinds- Base-Excision Repair and Nucleotide-Excision Repair  Base Excision Repair Used to replace chemically modified bases AP Site (apurinic/apyrimidinic site): a location in DNA that has neither a purine nor a pyrimidine base Four-step process Glycosylase – removes altered base (not the entire nucleotide) AP endonuclease and Phosphodiesterase– removes the remainder of the nucleotide Polymerization – fills the empty spot Molecular Biology of the Cell (© Garland Science 2015) Ligation – seals the nick in the DNA backbone  Nucleotide Excision Repair Recognizes general distortions caused by bulky Thymine Dimer chemical adducts –up to 30 bases long Bulky lesions/adducts include Pyrimidine dimers due to UV Radiation, chemotherapy, strand breaks, crosslink across chains DNA- protein crosslinks Covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene) Molecular Biology of the Cell (© Garland Science 2015) Exonuclease cuts out the defect, bases are restored, and the backbone is ligated In eukaryotes: 25+ enzymes  Nucleotide Excision Repair Exonuclease cuts out the defect, bases are restored, and the backbone is ligated In E. coli, UvrA, UvrB and UvrC remove the dimer induced by UV light In yeast, the proteins similar to Uvr's are named RADxx ("RAD" stands for "radiation"), such as RAD3 RAD10. Molecular Biology of the Cell (© Garland Science 2015) In eukaryotes, usually more than 25 enzymes are involved in this repair  Nucleotide Excision Repair mechanism related disease Human disease: Xeroderma Pigmentosum Rare autosomal recessive disorder DNA of skin cells damaged by UV light cannot be repaired Some cancers and neurologic problems can be life-threatening Lack of excision repair mechanism o XPA (DNA Damage Recognition and Repair Factor) gene defective mutation ▪ XPA protein binds to the damaged DNA ▪ XPA protein recruits other proteins to perform nucleotide excision repair  Double-Strand Break (DSB) Especially dangerous type of DNA damage Caused by Ionizing radiation, replication errors, oxidizing agents, and other metabolites produced in the cell cause breaks of this type Un-repaired DSBs will cause the breakdown of chromosomes into smaller fragments Two mechanisms to repair Double-Strand Break Non-homologous end joining (NHEJ) Homologous recombination (HR) Non-homologous end joining predominates in humans Homologous recombination is used only during and shortly after DNA replication when sister chromatids are available to serve as templates  Non-homologous End-joining (NHEJ) Three-step repair - Break is recognized by Ku heterodimers - The protein complex holds the broken end together and trims the ends of the breaks - The blunted ends are ligated A “quick and dirty” solution usually causes deletions Common in mammalian somatic cells An acceptable solution since so little of the mammalian genome is essential for life. Molecular Biology of the Cell (© Garland Science 2015) Chance to cause aberrant chromosomes - with two centromeres or no centromeres Homologous Recombination Exchange of DNA strands between a pair of homologous duplex DNA, which may be identical or similar HR occurs just during or right after DNA replication when two daughter DNA molecules lie close together, and one of them serves as a template One stretch of duplex DNA acts as a template to restore lost or damaged information on the second duplex stretch https://www.bartleby.com/ HR accurately repairs DSB Facilitates chromosomal crossover  HR repair DSB Nuclease digests the broken strands to release single-strand 3’ end Strand exchange/invasion: one of the single-strand 3’ ends worms its way into the template duplex and anneals to the complementary region Polymerase synthesizes DNA using the unbroken strands as a template to restore the damaged region Strand displacement: the invading strand is released, DNA synthesized and ligated to restore the two original DNA double helices. HR exploits a complementary strand from a separate DNA duplex Molecular Biology of the Cell (© Garland Science 2015)  HR is crucial for Meiosis Deliberately exchange of material between different chromosomes to generate novel combinations of genes HR produces chromosome crossing- over, resulting in hybrid chromosomes that contain genetic information from both the maternal and paternal homologs This exchanging of DNA is an important Molecular Biology of the Cell (© Garland Science 2015) source of the genomic variation seen among offspring  Human diseases related to defective HR ❖ Brca1 regulates an early step in broken-end processing; without it, such ends are not processed correctly for homologous recombination and instead are repaired inaccurately by the non-homologous end-joining pathway ❖ Brca2 binds to the Rad51 protein, preventing its polymerization on DNA, and thereby maintaining it in an inactive form until it is needed ❖ Brca1 and Brca2 protein mutations lead to a greatly increased frequency of breast and ovarian cancer Other Human Diseases Table 5-2 Molecular Biology of the Cell (© Garland Science 2015) Study questions 1. True or False: HR is the predominant mechanism for repairing double-strand breaks in human cells since it is more accurate than NHEJ. 2. True or False: Xeroderma pigmentosum is a disease caused by defects in base excision repair mechanisms. 3. True or False: BRCA1 and BRCA2 mutations are associated with an increased risk of breast and ovarian cancer due to their role in homologous recombination. 4. True or False: UV radiation primarily causes DNA damage by forming pyrimidine dimers, such as thymine-thymine (TT) or cytosine-cytosine (CC) dimers. 5. True or False: Homologous recombination (HR) can only occur during meiosis and is not used for DNA repair in somatic cells. Answers: 1: F; 2: F; 3: T; 4: T; 5: F.