Exam 4 Review Genetic Disorders.pdf

Central Dogma, DNA Synthesis, and Cell Cycle DNA Replication DNA synthesis occurs by the process of replication • DNA replication requires many proteins that are responsible for maintaining the separation of the parental strands, and for unwinding the double helix ahead of the replication fork. Ma...

Central Dogma, DNA Synthesis, and Cell Cycle DNA Replication DNA synthesis occurs by the process of replication • DNA replication requires many proteins that are responsible for maintaining the separation of the parental strands, and for unwinding the double helix ahead of the replication fork. Major players: • DNA Helicases – enzymes responsible for forcibly separating the DNA strands and unwinding the parental duplex • Single-stranded DNA binding proteins – prevent the strands from reassociating and protect the strands from enzymatic cleavage (Ex. RPA) • DNA Topoisomerases – enzymes that are responsible for removing positive and negative supercoils that form as a result of overwinding by transiently cleaving one or both strands of DNA [Ex. DNA gyrase (Bacterial topoisomerase)] • DNA polymerases synthesize the new DNA strands only in the 5’à3’ (antiparallel) direction. To ensure replication fidelity, some DNA polymerases have a “proofreading” activity (3’à5’ exonuclease) in addition to their 5’à3’ polymerase activity • DNA ligases catalyze the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA. Clinical Note-Bloom syndrome Bloom Syndrome is an autosomal recessive genetic disorder characterized by DNA Helicase deficiency (BLM mutation). • Growth retardation • Butterfly rash • Compromised immune system • Elevated risk of different cancers • Average lifespan ~25 years (BS; OMIM #210900; ORPHA #125) https://plasticsurgerykey.com/136-rothmundthomson-syndrome-bloom-syndromedyskeratosis-congenita-fanconi-anaemia/ DNA Damage and Repair Mechanisms • Damage to cellular DNA is involved in mutagenesis and the development of cancer • DNA in a human cell undergoes several thousand to a million damaging events per day, generated by both external (exogenous) and internal metabolic (endogenous) processes. Damage can affect single strands or both strands of DNA • Changes to the genome can generate error in the transcription and eventual translation into proteins necessary for signaling and cellular function • Once cells lose their ability to effectively repair damaged DNA, there are three possible responses: • Senescence or irreversible dormancy • Apoptotic b/c of damage triggered apoptotic signaling cascade • Malignant i.e., develop immortal characteristics and begin uncontrolled division To compensate for the degree and types of DNA damage that occur, cells have developed multiple repair processes 4 Sources of DNA Damage Environmental (exogenous) sources Chemical mutagen DNA alkylating agents (e.g., nitrogen mustards) Procarcinogens (polycyclic aromatic hydrocarbons(PAH) e.g., benzo[a]pyrenes(BaP)) Physical mutagen (primarily radiation sources) Ultraviolet light from the sun can cause pyrimidine dimers High-energy ionizing radiation (e.g., X-rays) can cause single or double strand breaks Endogenous sources (metabolic and biochemical reactions) Oxidation of bases due to reactive oxygen species (ROS) can lead to base damage, single strand breaks (SSB), or double stranded breaks(DSB). Loss of bases can also occur spontaneously or due to external factors, such as exposure to heat, acidic conditions, or chemicals. Genomic mutations introduced during DNA replication in the S phase Polymerase incorporates the wrong base Polymerase “stuttering” due to strand slippage when copying a DNA segments with many repetitive sequences- mismatches bases Clinical Note: Melanomas develop from exposure of the skin to the UV rays to the sun. The UV radiation causes pyrimidine dimers to form in DNA. Mutations may result from non-repair of the dimers that produce melanomas, appearing as dark brown growths on the skin. DNA Repair Steps Common Steps of DNA Repair 1. Damage recognition 2. Excision (removal of damaged DNA) 3. Repair (resynthesis) 4. Ligation 1: DNA Damage Recognition • MRN complex is a multi-protein complex involved in DNA damage sensing, repair, and signaling. • MRE11 (meiotic recombination 11)- nuclease activity involved in end processing of broken DNA strands • RAD50 (radiation-sensitive 50)- important for DNA end processing and DNA tethering • NBS1 (Nijmegen breakage syndrome 1)-scaffold protein that connects MRN complex. -MRN recruits many proteins involved in single and double strand DNA repair e.g., it activates ATM (Ataxiatelangiectasia mutated) • ATM phosphorylates several proteins involved in • Cell cycle arrest • DNA repair (mainly in double strand breaks) Single Stranded DNA Repair Mechanisms 3 Common Steps after damage recognition • Excision • Repair • Ligation 3 major mechanisms of Single Stranded DNA Repair • Nucleotide Excision Repair (NER) Subtypes of NER -Global genomic(GG) -Transcription-Coupled Repair (TCR) • Base Excision Repair (BER) • Mismatch Repair (MMR) Nucleotide Excision Repair (NER) Repairs: local distortions of DNA helix (e.g., pyrimidine dimers from UV radiation or bulky adducts from oxidized benzo[a]pyrene) Steps: • Specific repair endonucleases recognize and cleave the abnormal chain on both the 3’ and 5’ side of the distorted region • A short oligonucleotide is released containing the distortion, leaving a gap in the DNA • The gap is filled using DNA polymerase and DNA ligase TCR-NER sub-pathway removes DNA damage from actively transcribed genes GG-NER sub-pathway removes damage throughout the genome. Xeroderma Pigmentosum (XP) • Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder which is caused primarily by gene mutations in the GG-Nucleotide Excision repair pathway. These mutations impair the ability to repair damage caused by ultraviolet (UV) light. This condition causes freckle-like spots and is linked to high incidences of skin cancer. In extreme cases, all exposure to sunlight must be forbidden, no matter how small; as such, individuals with the disease are often colloquially referred to as Children of the Night. • Pyrimidine dimers occur frequently in the skin. • Deficiency of the endonucleases involved in the removal of pyrimidine dimers from DNA. • Because of the inability to repair DNA, the frequency of mutations increases. • The risk of developing skin cancer: • Non-Melanoma: 10,000X increased risk • Melanoma: 2000X increased risk Cockayne Syndrome (CS) Cockayne Syndrome is a rare autosomal recessive disorder linked to defects in nucleotide excision repair, specifically TCR-NER (transcription-coupled repair). Cockayne syndrome can results from mutations in with the ERCC6 gene or the ERCC8 gene which code for proteins involved with TCR. Mutations caused by UV irradiation, chemicals, free radicals, etc. build within the genes that are expressed and result in eventual cell malfunction and death. The increased cell death likely contribute to the features of growth failure and premature aging. Characteristics: • Sensitivity to UV irradiation (but NOT associated with skin cancer) • Failure to thrive (gain weight and developmental growth) • Appearance of premature aging • Impaired development of the nervous system • Hearing loss, eye abnormalities, severe tooth decay 12-year-old Chinese girl with CS Base Excision Repair (BER) Repairs: DNA lesions involving base alterations or spontaneous base loss (e.g., damage from nitrogen mustards, base oxidation, or deamination) Steps: 1. Removal of abnormal bases: • Abnormal bases are recognized by specific Glycosylases that hydrolytically cleave the base from the sugar-phosphate backbone • This leaves an apyrimidinic site or an apurinic site (both referred to as AP sites) 2. Recognition and repair of an AP site: • Specific AP Endonucleases recognize that a base is missing and initiate the process of excision by making an endonucleolytic cut on the 5’ side of the AP site • A deoxyribose phosphate Lyase removes the single, base-free, sugar phosphate reside • DNA Polymerase and DNA Ligase complete the repair process GEL PLease Mismatch Repair (MMR) Repairs: NON-damaged mismatched bases because of an error in DNA polymerase proofreading function or the result of a DNA polymerase slip or stutter MMR is mediated by a group of proteins known as the Mut proteins in bacteria [homologous proteins are present in humans: e.g., MLH1, MSH2, MSH6, PMS2] Steps: 1. Identification of mismatched stand • Prokaryotes: Discrimination of correct strand is based on the degree of methylation. GATC sequences, which are found every ~ 1000 NT, are methylated on the adenine (A) on parental strand • Eukaryotes: Strand selection is based on the nicks in the strands and how the Mut homologs interact with PCNA, which forms the leading edge of the replication fork. 2. Repair of damaged DNA • An endonuclease nicks the strand, and the mismatched nucleotides are removed by an exonuclease • The gap is filled by DNA polymerase and ligated to the original stand by DNA ligase 12 Mismatch Repair (MMR) • Microsatellites are regions of DNA sequences of 1-6 nucleotide base pairs repeated in tandem ~5-50X • These sites are sometimes called “canaries in the coal mine” -They tell us that mutations are also occurring elsewhere -MSI-is a hypermutable phenotype caused by the loss of DNA mismatch repair activity. Double-stranded break (DSB) repair • DNA double-stranded breaks are hazardous forms of DNA damage which can lead to genome rearrangements, mutations, and cell death • Two mechanisms by which the cell attempts to repair a complete break in a DNA molecule • Homologous recombination (HR) • Non –homologous end joining (NHEJ) • Defects in these repair mechanisms lead to variety of genetic diseases most of which include ionizing radiation sensitivity and chromosome aberrations 14 Non-Homologous End Joining (NHEJ) • A pathway that repairs double-stranded breaks in DNA by direct joining of broken ends • NHEJ is referred to as “non-homologous” because the break ends are directly ligated without the need for a homologous template • A protein Ku is essential for NHEJ as it recognizes and binds to the exposed ends for ligating • Imprecise repair leading to loss of nucleotides occurs frequently with NHEJ [without a validating DNA template present, the repair results in non-original DNA strand formation] • Errors in direct joining may be a cause of the various translocations that are associated with cancers (e.g., Burkitt’s lymphoma) 15 Burkitt’s Lymphoma Burkitt’s Lymphoma is a highly aggressive B- cell non-Hodgkin lymphoma cancer that is characterized by the translocation and deregulation of the c-myc on chromosome 8. The classic t(8;14) reciprocal translocation (85% cases) results in the transposition of the c-myc on chromosome 8 with one of the immunoglobulin heavy chain genes on chromosome 14, which results in activation of the c-myc gene and is considered responsible for tumor proliferation. Overproduction of the c-myc product may change the lymphocytes into cancer cells. C-myc is a leucine zipper transcriptional activator (proto-oncogene) that affects different pathways regulating cell cycle, growth, adhesion, differentiation, and apoptosis. It is overexpressed via its juxtaposition with immunoglobulin gene enhancers leads to a deregulation of cell cycle control. Burkitt’s lymphoma is one of the fastest growing malignancies in humans, with a growth fraction close to 100% (doubling time of around 25 hours). 16 Homologous Recombination (HR) • A form of homology-directed repair used to mend the broken ends of double-strand DNA lesions. • HR can only be used by the cell when there is a homologue piece of DNA present (information on the intact sister chromatid or on the homologous chromosome) • Assumed to be error-free because of the use of homologous DNA segments to restore damaged information • Two of the proteins, BRCA1 and BRCA2,have distinct yet interconnected roles in HR • Inherited mutations in these genes predispose women to breast and ovarian cancers Homologous Recombination defective: e.g., Breast and Ovarian Cancer Syndrome 17 Ataxia Telangiectasia Ataxia Telangiectasia is an autosomal recessive, complex, multisystem disorder that is due to a defect in the ATM kinase. ATM Kinase assists cells in recognizing damaged or broken DNA stands during homologous recombination and NHEJ which coordinates DNA repair by activating enzymes that fix the broken strands. ATM mutated cells fail to activate checkpoints in response to DNA damage, exhibit increased genomic instability at the chromosome level, and have an increased risk of lymphomas and/or leukemia's. Without this protein, cells become unstable and die (e.g., loss of brain cells that coordinate movement). AT cells and individuals are hypersensitive to ionizing radiation. Characteristics: • Ocular and cutaneous telangiectasia • Progressive difficulty with coordinating movements (ataxia) • Progressive neurologic impairment • Variable immunodeficiency with susceptibility to sinopulmonary infections • Impaired organ maturation 18