DNA Mutagenesis and DNA Repair Mechanisms

Andrea Byrum March 21, 2016 Overview

• Mutaonal signaturesà what are they? What can they tell us about the mutaonal process? • Types of DNA damage and the mutaons they cause – Endogenous DNA damage – Exogenous DNA damage • DNA repair processes • Double stranded breaks Mutaonal Processes

Helleday et al., 2014 Mutaonal Signatures

Helleday et al., 2014 Different forms of Damage make specific contribuons to a mutaonal signature

Helleday et al., 2014 Endogenous DNA Damage Endogenous DNA damage: deaminaon • occurs spontaneously in all DNA bases that contain primary amines

hps://biotechkhan.wordpress.com/tag/deaminaon/ Spontaneous deaminaon of 5meC at CpG dinucleodes

Helleday et al., 2014 Cytosine deaminaon to uracil • Catalyzed by cytosine deaminase family enzymes – AICDA (acvaon-induced cydine deaminase) • Funcons in anbody diversificaon – APOBEC (apolipo protein B mRNA eding enzyme, catalyc polypepde) • Inolved in mRNA eding, and defends against retrovirus or retrostransposon invasion

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Helleday et al., 2014 Endogenous DNA damage: Free Radicals • Include reacve oxygen species (ROS) and nitrogen oxide species (NOS) • Byproducts of normal cellular metabolism – Their interacons with DNA can cause >25 different oxidave base lesions – Ex: 8-Oxoguanine binds preferenally to adenine, resulng in GŸCà TŸA transversions

8-oxoG (syn) in a Hoogsteen base pair with dA (an) Endogenous DNA damage: DNA replicaon errors

• High-fidelity DNA polymerases δ and ε have an error rate of 1 in 10-7 • Mutaons resulng in proofreading defects dramacally increase replicaon errors • Balance of dNTPs can also affect accuracy

Mahews et al., 2015 Exogenous DNA Damage Exogenous DNA damage: UV radiaon

• High energy allows for covalent bonds to form between neighboring pyrimidine nucleodes Exogenous DNA damage: Chemical Mutagens

• Alkyang agents: cause CŸGàTŸA transions – Nitrogen mustards – methyl methanesulphonate (MMS) – N-nitroso-N-methylurea (NMU) – N-ethyl-N-nitrosourea (ENU) – Ethyl methanesulfonate (EMS) – Chemotherapeuc drugs (cyclophosphamide, temozolomide) Exogenous DNA damage: alkylang agents

Transions = mutaons that involve the same class of nucleodes (ie. Purine- to-purine) Exogenous DNA damage: chemical mutagens • Planum-based compounds: cause bulky adducts and crosslinking – Ex: cisplan (chemotherapy drug) • Psoralens: cause pyrimidine mutaons in TpA sequences; used to treat skin condions • Ionizing radiaon: causes double strand breaks and produces ROS • Intercalang agents: cause GŸCàTŸA transversions – Ex: Benzo[a]pyrene (carcinogen), daunorubicin, acnomycin-D (ancancer drugs) Benzo[a]pyrene Mutagenesis

Transversions=mutaons that involve different classes of nucleodes (ie. Purine-to-pyrimidine and vice-versa) DNA Repair Pathways DNA Repair Processes: Base Excision Repair (BER)

Removes bases (via oxidaon, alkylaon, or deaminaon) that could cause mutaons by mispairing or lead to breaks in DNA during replicaon DNA Repair Processes: Nucleode Excision Repair (NER) Transcripon-coupled repair

Acvated when bulky adducts are sensed in the DNA (ex: B[a]P and UV-induced lesions)

DNA Repair Processes: Mismatch repair (MMR)

Recognizes and repairs mis- incorporated bases and indels arising from DNA replicaon and DNA recombinaon repair Double Stranded Breaks (DSBs) • Primary DSBsà direct breaks in the sugar-phosphate backbone of the DNA molecule (ie. Ionizing radiaon, nucleases) • Secondary DSBsà result from DNA lesions that are encountered by a replicaon fork and the fork collapses DNA Repair Processes: Non- homologous end-joining (NHEJ) DNA Repair Processes: Homologous recombinaon (HR) DNA Repair Processes: Homologous recombinaon (HR)

Synthesis-dependent end-joining leads to tandem duplicaons Conclusions

• Mutaons that occur in nature or that are generated purposely in the lab differ depending on the parcular source of DNA damage and the repair pathway ulized to resolve it • Important to understand the effects of your mutagen when performing an experiment – Will you get the types of mutaons you want in your screen? – Will other types of damage that you are not studying be induced by your treatment?