Oxidative Stress and Repair Oxidative DNA modifications Faiez Al Nimer Oxidative lipid modifications Huang Liyue Oxidative protein modifications Sanyal Nikhilesh Oxidative DNA and protein damage repair Melanie Neely Willis

Oxidative DNA damage Reactive oxygen species Strand breaks Oxidized base adducts DNA-DNA and DNA-protein cross links Both nuclear and mitochondrial DNA is attacked by ROS but mtDNA is affected more because it does not contain histones it is in proximity to ROS generation and has limited antioxidant repair system

Oxidized base adducts: Hydroxyl radical Evans et al, Reviews in mutation research, 2004

8-OH-Gua and Thymin glycol Guanine: lowest oxidation potential  most oxidized base

Cell viability and disease  For 8-OH-Gua: Results in C→A and G→T transversions, leading to mutations Affects transcription factor binding and gene expression Interferes with methylation of i.e. oncogenes  For thymin glycol: T→C transitions have been noted Blocks replication

DNA oxidation: Neurodegenerative diseases 8-OHDG stainingp62 promoter oxidation Lovell et al, J Neurochem, 1999 Du et al, Neurobiol of disease,2009

DNA oxidation: Neurodegenerative diseases  Absence of p62 induces cell death  p62 protects cells from oxidative stress via the antioxidant response element (ARE) Du et al, Neurobiol of disease,2009 p62 expression

Oxidative Stress and Repair Oxidative DNA modifications Faiez Al Nimer Oxidative lipid modifications Huang Liyue Oxidative protein modifications Sanyal Nikhilesh Oxidative DNA and protein damage repair Melanie Neely Willis

Lipid modifications by reactive oxygen species (ROS) Overproduction of ROS damages cellular components, including lipids, leading to decline in physiological function and cell death

Reaction of ROS with lipids lead to Lipid oxidation Lipid peroxidation

Lipid Oxidation Initiation – A radical is formed Propagation – Radicals react and transfer their unpaired electron to other compounds Termination – Two radicals combine to stop the reaction

Adibhatla RM; Hatcher JF. Antioxid Redox Signal. 2010

Lipid peroxidation LH OH or M=O L L + O 2 LOO LOO + LH L + LOOH LOOH + metal Initiation Propagation LO LOO Hydroperoxide Peroxy radical Alkoxy radical Alkanes Malondialdehyde 4 hydroxynananol 8-isoprostanes Commonly measured decomposition products

Adibhatla RM; Hatcher JF. Antioxid Redox Signal. 2010

Oxidative Stress and Repair Oxidative DNA modifications Faiez Al Nimer Oxidative lipid modifications Huang Liyue Oxidative protein modifications Sanyal Nikhilesh Oxidative DNA and protein damage repair Melanie Neely Willis

Effects of Reactive oxygen species on Protein backbone Oxidation of proteins often targets them for degradation (20S proteasome and other proteases). Several age-related disorders are characterised by Protein oxidation. E.g. Parkinson’s disease, Amyotropic Lateral sclerosis. Product of the oxidation (Peptide alkoxyl radical) is susceptible to cleavage by diamide or  -amidation pathways. Redox Biochemistry, Wiley Interscience, 2008; p186

Oxidation of amino acid residue side chain Redox Biochemistry, Wiley Interscience, 2008; p188

Oxidation of amino acid residue side chains : Tyrosine Larios J M et al. J. Biol. Chem. 2001;276: Qun Gao C et al. Cardiovasc Res 2003;57: Tyrosine oxidation can lead to dityrosine formation. This leads to protein crosslinking. Useful Biomarker for Oxidative damage and aging. Increased levels of dityrosine is found in rat hearts where Matrix metalloproteinase-2 (MMP-2) was induced by cytokines. Tyrosine Dityrosine

Oxidation of amino acid residue side chains : Cysteine Green J and Paget MS (2004) Nat Rev Microbiol. 2(12): R’ - Glutathione or another protein Sulfonic acid Sulfinic acid Sulfenic acid

Beneficial effects of ROS on Proteins : e.g. DJ-1 H 2 O 2 oxidises the Cys106 (-SH) to sulfinic acid (-SOOH) Blackinton et al et al. J. Biol. Chem. 2009;284: Oxidised Cys106 Partially oxidised Cys106 (Sulfenic acid) Cys106 oxidation is controlled by its neighbouring environment.

Beneficial effects of ROS on Proteins : e.g. DJ-1 Cys106 is essential for maintaining nuclear morphology. Shown here are M17neuroblastoma cells Cys106 helps increase Cell viability under mitochondrial stress Blackinton et al et al. J. Biol. Chem. 2009;284:

Oxidative Stress and Repair Oxidative DNA modifications Faiez Al Nimer Oxidative lipid modifications Huang Liyue Oxidative protein modifications Sanyal Nikhilesh Oxidative DNA and protein damage repair Melanie Neely Willis

DNA & Protein Repair Enzymes Oxidation of pyrimidine and purine bases leads to DNA damage. Oxidation of methionine and cysteine residues can lead to protein damage and aggregation. DNA damage is repaired by several enzymes in the Base Excision Repair pathway (BER). Proteins are repaired by methionine sulfoxide reductases (MsrA, MsrB) and sulfiredoxins.

Base Excision Repair 1.Removal of the damaged base from the sugar by a damage-specific glycosylase 2.Processing of the apyrmidinic-apurinic (AP) site by an AP endonuclease and deoxyribosylphosphate lyase. 3.Replacement of an undamaged nucleotide by DNA polymerase. 4.Repairing nicks in the DNA backbone by DNA ligase.

Crystal Structure Shows DNA Bending Lukianova OA, David SS. A role for iron-sulfur clusters in DNA repair. Curr Opin Chem Biol. 2005;9:145–151.

Reversibility of Prx Hyperoxidation Before exposure After 10 minutes exposure to H Recovery for 4 hours 10 more minutes of exposure to H Recovery for 8 hours Woo HA, Chae HZ, Hwang SC, Yang KS, Kang SW, Kim K, Rhee SG.Science Apr 25;300(5619):592-4.

Mechanism of reduction of Prx-SO2− by Srx. Jönsson T J et al. J. Biol. Chem. 2009;284: ©2009 by American Society for Biochemistry and Molecular Biology