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DNA Mutagenesis and DNA Repair Mechanisms
Andrea Byrum March 21, 2016
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Overview
• Muta?onal signaturesà what are they? What can they tell us about the muta?onal process?
• Types of DNA damage and the muta?ons they cause – Endogenous DNA damage – Exogenous DNA damage
• DNA repair processes • Double stranded breaks
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Muta?onal Processes
Helleday et al., 2014
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Muta?onal Signatures
Helleday et al., 2014
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Different forms of Damage make specific contribu?ons to a muta?onal signature
Helleday et al., 2014
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Endogenous DNA Damage
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Endogenous DNA damage: deamina?on
• occurs spontaneously in all DNA bases that contain primary amines
hOps://biotechkhan.wordpress.com/tag/deamina?on/
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Spontaneous deamina?on of 5meC at CpG dinucleo?des
Helleday et al., 2014
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C U
Cytosine deamina?on to uracil • Catalyzed by cytosine deaminase family enzymes
– AICDA (ac?va?on-‐induced cy?dine deaminase) • Func?ons in an?body diversifica?on
– APOBEC (apolipo protein B mRNA edi?ng enzyme, cataly?c polypep?de) • Inolved in mRNA edi?ng, and defends against retrovirus or retrostransposon
invasion
Helleday et al., 2014
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Endogenous DNA damage: Free Radicals
• Include reac?ve oxygen species (ROS) and nitrogen oxide species (NOS)
• Byproducts of normal cellular metabolism – Their interac?ons with DNA can cause >25 different oxida?ve base lesions
– Ex: 8-‐Oxoguanine binds preferen?ally to adenine, resul?ng in G�Cà T�A transversions
8-‐oxoG (syn) in a Hoogsteen base pair with dA (an%)
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Endogenous DNA damage: DNA replica?on errors
• High-‐fidelity DNA polymerases δ and ε have an error rate of 1 in 10-‐7
• Muta?ons resul?ng in proofreading defects drama?cally increase replica?on errors
• Balance of dNTPs can also affect accuracy
MaChews et al., 2015
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Exogenous DNA Damage
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Exogenous DNA damage: UV radia?on
• High energy allows for covalent bonds to form between neighboring pyrimidine nucleo?des
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Exogenous DNA damage: Chemical Mutagens
• Alkya?ng agents: cause C�GàT�A transi?ons – Nitrogen mustards – methyl methanesulphonate (MMS) – N-‐nitroso-‐N-‐methylurea (NMU) – N-‐ethyl-‐N-‐nitrosourea (ENU) – Ethyl methanesulfonate (EMS) – Chemotherapeu?c drugs (cyclophosphamide, temozolomide)
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Exogenous DNA damage: alkyla?ng agents
TransiIons = muta?ons that involve the same class of nucleo?des (ie. Purine-‐to-‐purine)
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Exogenous DNA damage: chemical mutagens
• Pla?num-‐based compounds: cause bulky adducts and crosslinking – Ex: cispla?n (chemotherapy drug)
• Psoralens: cause pyrimidine muta?ons in TpA sequences; used to treat skin condi?ons
• Ionizing radia?on: causes double strand breaks and produces ROS
• Intercala?ng agents: cause G�CàT�A transversions – Ex: Benzo[a]pyrene (carcinogen), daunorubicin, ac?nomycin-‐D (an?cancer drugs)
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Benzo[a]pyrene Mutagenesis
Transversions=muta?ons that involve different classes of nucleo?des (ie. Purine-‐to-‐pyrimidine and vice-‐versa)
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DNA Repair Pathways
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DNA Repair Processes: Base Excision Repair (BER)
Removes bases (via oxida?on, alkyla?on, or deamina?on) that could cause muta?ons by mispairing or lead to breaks in DNA during replica?on
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DNA Repair Processes: Nucleo?de Excision Repair (NER)
TranscripIon-‐coupled repair
Ac?vated when bulky adducts are sensed in the DNA (ex: B[a]P and UV-‐induced lesions)
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DNA Repair Processes: Mismatch repair (MMR)
Recognizes and repairs mis-‐incorporated bases and indels arising from DNA replica?on and DNA recombina?on repair
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Double Stranded Breaks (DSBs) • Primary DSBsà direct breaks in the sugar-‐phosphate backbone of the DNA molecule (ie. Ionizing radia?on, nucleases)
• Secondary DSBsà result from DNA lesions that are encountered by a replica?on fork and the fork collapses
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DNA Repair Processes: Non-‐homologous end-‐joining (NHEJ)
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DNA Repair Processes: Homologous recombina?on (HR)
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DNA Repair Processes: Homologous recombina?on (HR)
Synthesis-‐dependent end-‐joining leads to tandem duplica?ons
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Conclusions
• Muta?ons that occur in nature or that are generated purposely in the lab differ depending on the par?cular source of DNA damage and the repair pathway u?lized to resolve it
• Important to understand the effects of your mutagen when performing an experiment – Will you get the types of muta?ons you want in your screen?
– Will other types of damage that you are not studying be induced by your treatment?