Fate of induced DNA damage genotoxic agent>>> cell death or DNA damage>>> DNA repair>>> 2 types: error free repair OR error prone repair>>> point mutation OR chromosome mutation  

Exogenous Agents = UV light, chemicalsEndogenous Agents = ROS, RNSInefficient DNA repair - Mutation in tumor suppressor genes / oncogenes-Chromosomal instability & Cancer Efficient Genome Safeguarding System (DNA repair)-Genomic Stability

Pathway Known to be activated following DNA damage- DNA repair- Cell cycle Checkpoints- Transcription- Apoptosis (programmed cell death)

Apoptosis: KEY FEATURES-Cell Shrinkage -Loss of surface contact with neighbours -Chromatin condensation around edge of nucleus(or fragmentation into small balls of condensed chromatin)-Fragmentation of the apoptotic cell into small apoptotic bodies (contents not leaked out)-Phagocytosis by neighbour/macrophage 

Cell Death, DNA Repair and Diseases  

DYSREGULATED APOPTOSIS IN DISEASEIncreased1. AIDS & Other viral diseases2. Ischemic brain damage3. Neurodegenerative disorders (e.g retinal degeneration, parkinson's, alzheimer'sDecreased1. Developmental disorders eg ADHD2. Autoimmune diseases eg Graves3. Inflammatory4. Carcinogenesis (over expression of anti-apoptosis gene BCL 2; loss of functional gene)

Why Study DNA Repair Processes?-Repair occurs by many different biochemical strategies-Repair genes account for a significant fraction of genome-DNA repair defects lead to genetic instability, which is a hallmark of cancer-Genetic instability in cancer cells allows them to become resistant to treatment  

A diversity of DNA lesions= A diversity of DNA repair mechanisms Genotoxic agents include: UV, PAHs, oxidative stress, ionizing radiations, drugs etc.  1. Base Excision Repair>>> BER is initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases, forming AP sites. These are then cleaved by an AP endonuclease. The resulting single-strand break can then be processed by either short-patch (where a single nucleotide is replaced) or long-patch BER (where 2-10 new nucleotides are synthesized). 2. Nucleotide Excision Repair 3. Mismatch Repair *3 pathways share common « cut and paste » mechanisms  (excision/synthesis)  4. Direct Reversion 5. Recombination (HR & NHEJ)Homologous Recombination=1. Repairs DNA before cell enters M phase shortly after DNA replication in S and G2 phase where sister chromatids are easily visible2. Homologous sequence required to guide repair 3. Requires homologous sequences/chromosomes ;     Two primary models: DSBR pathway= double holiday junction model and SDSA= synthesis dependant strand annealing pathway4. Inappropriate HR leads to production of CABS>>> which are involved in carcinogenesisNon-Homologous End Joining (NHEJ)1. Predominant in G1 phase, when cells are growing but not yet ready to divide, (less frequent after G1 but maintains some activity through out the cell cycle)2. The break ends are directly ligated without the need for homologous template 3. Utilizes short homologous DNA sequences called 'microhomologies' often present in single stranded overhangs on the ends of dsb's 4. Inappropriate NHEJ can lead to translocation and telomere fusion>>> hallmarks of cancer  

Diversity of DNA repair pathways >>> Interacting NetworkComplexity, Redundancy, Coordination, Interplay 

Cell Cycle Checkpoints:- G1 Checkpoint >>> Is cell big enoughIs environment favorable - G2 CheckpointIs all DNA replicatedIs environment favorableIs cell big enough- Metaphase CheckpointAre chromosomes aligned on spindle

DNA Repair genes involved in Genomic InstabilityGene>>> ATM Aataxia telangiectasia mutated Protein kinase, recruited & activated by DNA double strand break's; activates DNA damage checkpoints Gene>>> CHK2 Checkpoint kinase 2 Protein kinase, activated in response to DNA damage, involved in cell cycle arrest, apoptosisGene>>> ATR Ataxia telangiectasia & Rad 3Protein kinase, sensing DNA damage (ssbs), activates DNA damage checkpoints BRCA 1, 2>>> Breast cancer susceptibility gene 1,2 Several functional domains interacting with tumour supressor genes and cell cycle progression.     

DNA repair processes (a) Photoreactivation (b) Specific removal of alkyl groups(c) base excision repair (d) nucleotide excision repair(e) post replication repair 

(a) Photoreactivation -Not in human-Not in dark-Major line of defence in plants -against UV induced damage (pyrimidine dimers & 6-4 photoproducts)

UV-induced Skin CancerFocus: UV radiation from sunlight Categories of sunlight -UVA-320-400 nm-UVB-290-320 nm-UVC - UV radiation, particularly at lower wavelengths has been associated with skin cancers 

UV and Skin Cancer  -UV radiation causes cancer by damaging DNA-Damage to the DNA caused by: Direct DNA damage (CPDs and 6-4PP)Oxidative stress-Most common damage leading to carcinogenesis involves the P53 tumour supressor gene -The other mechanisms of carcinogenesis  ~Immunosupression ~Inflammatory response  

Cyclobutane Pyrimidine Dimers (CPDs) >UVB photons give energy to dipyrimidine sites  >This leads to development of CPDs>The cytosine bases are unstable in in this configuration and deaminate, resulting in Uracil bases in their place >Translesion DNA synthesis(TLS) polymerases (particularly point) attempt to repair the DNA, however uracil bases can get replaced by thymine  >This results in UV "Signature" C>T mutation   

6-4PP and Dewar isomers -Another means of direct DNA damage is through the formation of 6-4 photoproducts which, under UVA radiation, become a Dewar isomer-Similar to CPDs, they cause mutations through mistaken repairs -Both 6-4PP and Dewer isomers are more easily repaired than CPDs and therefore cause less mutagenicity

Removal of alkyl groups-NH2 -OH & -SH groups in the DNA/ protein undergo alkylation (methylation)-Alkyl transferase enzymes transfer alkyl groups to one of its own cystine groups -Efficiency dependent upon available alkyl transferase molecules 

Base Excision Repair (BER)Evolved to protect cells from the damaging effects to DNA bases by spontaneous hydrolysis or ROS- Key enzyme = DNA glycosylases Different glycosylases to remove distinct varieties of damaged bases-Glycosylases cleave the bonds between bases & deoxyribose residues (i.e N glycosidic bonds) generating AP sites 

Some DNA Glycosylases = Thymine glycol DNA glycosylase    8-oxo-guanine/ Fapy DNA glycosylase   3-methyl adenine/ 3-methyl guanine    7-methyl guanine/ hypoxanthine DNA glycosylase    Formyl uracil DNA glycosylase    Uracil glycosylase     Fapy formamidopyrimidine 

Nucleotide Excision Repair (NER)-DNA removed as part of an oligonucleotide fragment -Complex, 30 different enzymes/proteins involved-Photodimer, oxidative damage PROCESS =>Breaking of phosphodiester bond on either side of the damaged site on the same strand >Gap filled by DNA repair synthesis >DNA ligase seals the remaining break

POST-replication recombinational repair  1. Replication blocked by pyrimidine dimer   2. Polymerase reinitiates downstream of dimer 3. Gap opposite dimer in daughter strand 4. Undamaged parental strand recombines into gap5. Recombination leaves a gap in previously intact parental strand 6. Gap filled by polymerase and ligase 

DNA REPAIR: - Preferential repair in active compared to inactive genes - Preferential repair in transcribed compared to non transcribed strand of active genesGBR global genome repair: Occurs throughout the genome including non-transcribed strand of active genesTranscription coupled repair TCR:Preferentially in the transcribed strand of transcriptionally active genes   

DNA Damage/Repair glossaryALKYLATIONAPURINICAPYRIMIDINIC DEAMINATIONDEPURINATIONEXCISIONPHOTOREACTIVATION

DNA repair deficient syndromes/Chromosomal breakage syndromes 1. XERODERMA PIGMENTOSUM2. ATAXIA TELANGIECTASIA3. FANCONI'S ANAEMIA 4. BLOOM SYNDROME 

1. XERODERMA PIGMENTOSUM= AUTOSOMAL RECESSIVE (1/240,000 births)Seven complementation groups (defects in 7 different genes involved in NER / BER)Clinical features: Sun sensitivity (UV radiation), dermatoses, hyperpigmentosum, retinal degeneration, neurological abnormalities, mental retardation, cancer 

2. Ataxia Talangiectasia = Autosomal recessive inheritance(1/ 100,000 births) First signs>>> Usually 2nd year of life, lack of balance and slurred speech, Progressive degenerative disease>>> characterised by cerebellar degeneration, ocular & cutaneous telangiectasia, immunodeficienc. radio sensitivity (e.g x rays) and predisposition to cancer Mutation in ATM gene (chromosome 11) leading to impairment to minimise DNA damage through cell cycle progressionPathway evolutionary conserved (also found in yeast) hence likely to be central to DNA damage response   

3. Fanconi's Anaemia = Autosomal Recessive in inheritance (2.5/ 10-5) Growth retardation (70 %), skin abnormalities, progressive bone marrow failure, neoplastic (cancer) riskMean age of onset>>> 8 years Mean age of det>>> 16 yearsSpontaneous chromatid/chromosomal breaks, hypersensitivity to clastogenic effects of DNA cross linking agents (e.g mitomycin C: MMC) used for diagnosis 7 Complementation groups! 

BLOOM Syndrome (BS)= Autosomal recessive, (frequency 2/10^5 newborns in ASHKENAZI JEWS and in JAPANESE) affected persons decent from a common ancestor; much rarer otherwise; 168 cases registered by James germanGrowth: dawarfism, intrauterine growth retardation, hyperpigmented spots on the skin, sun sensitive, butterfly rash across the face; prominant nose and / ears, normal intelligence; high risk of cancer.Chromosome /Chromatid type aberrations; highly elevated spontaneous SCEs rates Gene (BLM) located on Chromosome 15 makes protein consisting 1417 amino acids, accumulates high in S phase persists in G2/M and sharply declines in G1 Probable role in DNA replication (helicase) and DNA dsb repar 

10 HALLMARKS OF AGING~Genomic instability~Telomere attrition~Mitochondrial dysfunction~Cellular senescence ~Epigenetic alterations~ Loss of proteostasis~ Desregulated nutrient sensing~Stem cell exhaustion~Altered intracellular communication~Inflammation could be the 10th

Cell death, DNA repair

DNA Repair

Diseases