Lecture 8 DNA Damage repair and Cancer 1 Understand the difference between transition and transversion mutation A transition mutation occurs when one purine base becomes mutated or switched to another purine A Pyrimidine turning into a pyrimidine is also a transition mutation A transversion mutation is a purine and a pyrimidine becoming interchanged 2 Know the different types of DNA damages Endogenous Damage Replication errors mismatches Spontaneous loss of nucleosides base alteration chemical exposure base alteration Exogenous damage Chemical exposure base alteration UV light exposure thymine dimer Ionizing radiation double helix break 3 Understand the cellular response to DNA damages in cell cycle Role of Rb and E2F in S phase delay p53 p21 axis Our cells have many different responses to cell damage These include Cell Cycle Checkpoint Activation Damage Tolerance Dna Repair and Apoptosis Cell Cycle Checkpoint Activation This is a process that pauses the cell cycle in order to give the cell time to repair the damage before dividing and multiplying any more The cell cycle starts at G0 which is a stimulus which then goes to Growth and metabolism G1 then to DNA Replication S and then onto Preparation for cell division G2 and finally to Mitosis M This checkpoint can occur in state G1 during growth and metabolism or in G2 preparation for cell division In other words this cell cycle check can occur immediately before or after mitosis In the G0 and early G1 phases of the cell cycle pRB retinoblastoma protein physically associates with E2F transcription factor factors and blocks their transactivation domain Then in late G1 PRb releases E2F which allows the expressions of genes that encode for products necessary for the S phase Also ATM Ataxia Telangiectasia Mutated is involved in the process of converting P53 into activated p53 into p21 which is a precursor for a compound that stops the G1 step 4 Understand the differences of direct reversal repair base and nucleotide excision repair mismatch repair Direct Reversal Repair This is the most simple repair system and only involves bases In this process cells can eliminate damage to their DNA by chemically reversing it This does not require a template because this is a repair system that only manages repairing of base damage And thus this mechanism does not include the breakage of the phosphodiester minds In particular methylation of guanine bases is revered by a protein called MGMT Methyl Guanine Methyl Transferase in an alkylation reaction The next three are types of DNA repair which have three common steps 1 Recognize the offending base s 2 Remove the offending base s and 3 repair the resulting gap with a DNA polymerase and DNA ligase Base excision repair type of DNA repair BER This system corrects DNA damage from oxidation deamination and alkylation It creates a nick in the phosphodiester backbone of the AP site that was made when DNA glycosylase removes the damaged base Apurinic apyrimidinic AP endonuclease is an enzyme that is involved in the DNA BER pathway This system repairs damaged Deaminated alkylated DNA 8 oxo deoxyguanosine 3 methyladenine deaminated bases An example enzyme series Glycosylase to remove the wrong base AP endonuclease to prep the DNA for a new base And DNA polymerase and ligase to seal the nick with a new base Nucleotide excision repair type of DNA repair This system identifies the mismatched or mutated DNA strand nicks the mismatched mutated DNA Strand with endonuclease and removes it with an exonuclease This creates a gap Then a 5 3 DNA polymerase fills the gap and DNA ligase repairs the phosphodiester bond linkages to the repaired section Mismatch repair type of DNA repair AM I MISSING SOMETHING 5 Understand the pyrimidine dimer formed by ultraviolet light and the cause of xeroderma pigmentosum Exposure to UV light can cause adjacent thymines to form cross linkages which forms a thymine dimer This disrupts normal base pairing To fix this problem the body must identify the mutated DNA strand send an enzyme like excision endonuclease to cut out and remove the damaged DNA Then 5 3 DNA polymerase fills the gap and DNA Ligase forms the phosphodiester links Some people have a mutation in their excision endonuclease enzyme and they cannot eliminate thymine dimers This makes them highly susceptible to this kind of DNA damage and must stay out of the sun as much as possible People with this condition are said to have Xeroderma Pigmentosum or XP It is a rare autosomal recessive disease that is due to the deficiency of exi nuclease This was the first disease recognized to be caused by a defective DNA repair system Symptoms of this condition are photosensitivity high likelihood of skin cancer in sun exposed areas severe sunburns and blistering Skin is the most exposed area of the body to sunlight meaning that skin diseases are most common for someone with XP because more dimers are formed there XP leads to multiple basal cell carcinomas and other skin malignancies at young ages 6 Relate defective DNA repair to cancer xeroderma pigmentosum hereditary nonpolyposis colorectal cancer Familial Breast and Ovarian cancer their causes and relationships to DNA repair Roughly 80 of cancer cases are caused by environmental and food carcinogens This usually involves damage to DNA which induces SOS responses in the cell cycle Different problems in DNA repair and synthesis can result in various forms of cancer Xeroderma Pigmentosum or XP is related to the body s inability to correct thymine dimers in DNA There are 8 genes that this affects and it can lead to UV induced skin cancer The Nucleotide excision repair system is affected because genes that are affected in this syndrome are involved in the NER system hereditary nonpolyposis colorectal cancer or HNPCC This syndrome affects 4 5 genes and can result in colonic polyposis cancer The process affected is mismatch repair enzymes Familial Breast and Ovarian cancer This affects BRCA1 and BRCA2 genes and can result in breast and ovarian carcinomas The Homology directed repair of dsDNA breaks is the affected system 7 Understand the role of cytochrome P450 enzymes in activating carcinogens and mutagens Cytochrome P450 can convert procarcinogens to carcinogens This P450 compound takes aflatoxin B1 and converts it into aflatoxin B1 8 9 oxide which is an alkylating reagent Rat liver lysate is a source of cytochrome p450 which is a monooxygenase 8 How does Ames test work to identify