!Main Ideas - DNA damages causes cancer and aging, but also essential for evolution and genetic diversity!- Repairable DNA damage: cells activate survival response network!- Irrepairable DNA damage: causes mutagenesis (cancer?) or apoptosis!- DNA can be damaged spontaneously by various endogenous and exogenous mechanisms!- most common: single and two-base alterations, and DNA double strand breaks!- Major DNA repair pathways: MMR (replication errors), BER (single base changes), NER (2 or more nucleotide changes, DNA DSB repair!- Inherited mutations in DNA repair genes--> cancer predisposition and again!- Drugs that target DNA repair or DNA synthesis (replication) are often used in chemotherapy!- Chemotherapy targets fast dividing cancer cells, but also quickly dividing normal body cells (--> lots of side effects)!- Urgently needed= new strategies to selectively kill cancer cells Lecture Objectives 1) Describe the types of DNA damage 2) Discuss consequences of unpaired DNA damage 3) Explain 4 most common DNA repair mechanisms 4) Identify diseases caused by inherited DNA 5) Describe the different chemotherapeutic agents and their mode of action 1) Describe the types of DNA damage - Exogenous sources= UV and other radiation, chemicals!- Endogenous sources= ROS, alkylation, hydrolysis!- Spontaneous DNA alterations!- oxidative damage, hydrolytic attack, uncontrolled methylation, depurination, deamination!- Single base alterations!- Oxidation: caused by reactive oxygen species (ROS)--includes both radical and non-radicals!- Ex. 8-oxo-dG!- alkylation (methylation)!- crucial for devel. and assoc. with cellular processes (CG islands, gene repression, epigenetic inheritance), but it's dangerous because if you methylated cytosine and the deaminate it, you get thymine!- depurination: take off the whole base!- deamination: creates alternative nucleotides !- A--> hypoxanthine!- G--> xanthine!- C--> uracil!- T--> no deamination !- nucleotide insertion or deletion!- DNA adducts: when chemical groups are covalently attached to the DNA--> can intercalate with DNA double helix-->interfere with replication and transcription!- base annalog incorporation: when you add the structural analog of a base (ex. Bromouracil [BU] can bp with Guanine!- Alterations affecting 2 or more bases- Pyrimidine dimers: often caused by UV light. Covalent link between 2 T's or 2 C's on the SAME DNA strand. Block DNA replication and need a special DNA translesion polymerase to get across!- DNA crosslinks (intra and inter-strand)!- Interstand: blocks DNA replication, must be removed!- Intrastrand: blocks DNA repl, but can be passed by translesion polymerase!- induced by bifunctional alkylating and alykating-like agents (ex. sulfur mustard...once used for chemo)!- DNA double strand breaks!- DNA double strand breaks - considered the most deleterious type of DNA damage!- can cause translocations!- caused by ionizing radiation (gamma and x rays) or oxidative free radicals!- need DNA repair proteins! 2) Discuss consequences of unpaired DNA damage - Oxidation of nucleotides causes mutations (oxoG-->G-T conversion in repl.--> get T bped with A)!- Oxo-dG is marker for oxidative stress and the levels increase in the lung in response in inhaling fine particles. Increase assoc. with cancer, cardio diease, Alzheimer's - Deamination and depurination mutate the DNA!- DNA damage is really frequent!!- Cancer: DNA damage is the primary cause of cancer!- cancer incidence increases with age, mutations accumulate and DNA repair efficiency goes down as you age!- Aging!- Evolution!- Genetic diversity 3) Explain 4 most common DNA repair mechanisms - Cells have many DNA repair pathways, each has own enzyme set and "expertise"!- each mech. is essential (lose it-->cancer)!- most pathways highly conserved aka very important!- most precise pathways use undamaged DNA strand (homolog. chrom., sister chromatid) as a template!- Base excision repair - repairs depurination, deamination, oxidation, alkylation!- DNA glycosylases recognize and then remove the altered base!- AP (apurinic endonuclease and phosphodiesterase) remove the sugar-phosphate backbone!- DNA poly and DNA ligase fill the gap- Nucleotide excision repair - repairs mostly pyrimidine dimers!- a large complex recognizes the distorted DNA double helix!- an endonuclease clips the DNA and a helicase unwinds it and removes the damaged fragment !- DNA poly and ligase fill the gap - DNA double strand break repair - Homolog. Recomb.--> you will get a holiday junction--> can lead to crossover - Mismatch repair- muts and mut l 4) Identify diseases caused by inherited DNA - BRCA2: pheno= breast, ovarian, and prostate cancer. Enzyme/process affected= repair by homologous recombination.!- Fanconi anemia (what she works on) groups A-G: pheno= congenital abnormalities, leukemia, genome instability. Enzyme/process affected= DNA interstrand cross-link repair.!- Diseases in the chart are cancer-predisposition diseases. People with the defect in their inherited DNA are cancer prone.!- Xeroderma pigmentosum: defects in NER, UV light hypersensitivity, skin cancer!- Werner Syndrome: premature aging and cancer, defective DNA helicase that's involved in genome stability and telomere maintenance 5) Describe the different chemotherapeutic agents and their mode of action - the race between killing the cancer and killing the patient!- cancer cells divide the most rapidly, so you can kill them more effectively, but there are other human cells that also divide quickly (bone marrow cells, hair cells, cells of the GI tract).!-->common side effects of chemo: myelosuppresion (decreased production of blood cells), alopecia (hair loss), mucositis (inflammation of the lining of the digestive tract)!- Alkylating agents- induce DNA crosslinks - Antimetabolites- interfere with DNA synthesis - Anthracyclines (antibiotic)- DNA intercalators, block DNA rep or transcription - Plant alkaloids- MT inhibitors, block mitotic or meiotic cell division - Topoisomerase inhibitors- inhibit replication and cause DNA
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