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Mutation and DNA repair
1) DNA mutations can occur spontaneously from errors in replication or due to environmental mutagens, and can lead to genetic disorders if unrepaired. 2) The cell has multiple DNA repair mechanisms to recognize and fix damage, such as base excision repair and nucleotide excision repair. Failure to repair can increase mutations. 3) The study found that DNA damage increased phosphorylation and nuclear localization of the tumor suppressor PTEN, which plays a role in DNA repair and maintaining genomic integrity. Phosphorylation of PTEN was associated with enhanced DNA repair.
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Mutation and DNA repair
- 1. Mechanisms of Mutation AndDNA repair Thet Su Win MTMT/D-5736940
- 2. DNA and itsmutation • Components of DNA has its unique structures. • Its integrity and stability are essential for life. • Some factors can influence on DNA, resulting DNA damage. • If not repaired, will lead to mutation and possibly disease. • Permanent change in the DNA sequence is called Mutation. Clancy, S. (2008) DNA damage and repair: mechanisms for maintaining DNA integrity. Nature Education 1(1):103
- 3.
- 4. Spontaneous Mutations • Arisefrom errors in replication process and base modifications • Due to natural (biological) chemical processes • Base modificatons (spontaneous lesions) – Depurination – Deamination • Errors in DNA replication – Base substitution – Base insertion and deletion
- 5. Depurination • Loss ofa purine base, occurs spontaneously • A mammalian cell loses about 10,000 purines in 20hrs cell generation period
- 6. Depurination produces anApurinic site http://www.nature.com/scitable/content/depurination-produces-an-apurinic-site-55400
- 7. Deamination Deamination of (a)Cytosine and (b) 5-methylcytosine Griffiths et al (2004) (5-methyluracil)
- 8. Errors in DNAreplication Base Substitutions • No chemical reaction is perfect • Error in replication can occur when an illegitimate nucleotide pair (e.g. A-C) forms in DNA synthesis. • leading to a base substitution.
- 9. Base insertion anddeletion Griffiths et al (2004) Deletion Insertion Indel mutation
- 10. Induced Mutations • Ariseafter treatment with mutagens or environment agents. • Chemicals – Alkylating agents (ENU) – Methylating agents (EMS) – Polycyclic hydrocarbons – DNA intercalating agents (ethidium bromide) – DNA crosslinker (platinum) – Oxidative damage (oxygen radicals)
- 11.
- 12. Induced Mutations (Cont’d) •Radiation – UV (nonionizing radiation) – Ionizing radiation • Other mutagens – aflatoxin B1 (AFB1)
- 13. Classification of Mutations Mutations Small-scale mutations (microalteration) PointMutations Transition and Transversion Silent mutation Missense mutation Nonsense mutation Insertions Deletions Large-scale mutations (macroalteration) Amplification or duplication Deletions Inversions Rearrangement
- 14. Mutation and geneticdisorders Progeria • Autosomal recessive disorders • rapid aging disease • caused by single point mutation (in LMNA gene) • Thin, bald, old-looking skin
- 15. Mutation and haematological disorders •Sickle cell anaemia • a result of single nucleotide polymorphism (SNP) • Hb S http://evolution.berkeley.edu/evolibrary/article/mutations_06
- 16. Mutation and Cancer •Mutation in p53 tumor suppressor gene is the most prevalent mutation found in human cancers (over 50%) • P53 function – cell cycle regulation and apoptosis induction
- 17. Mutated p53 andcancer https://voer.edu.vn/m/cancer-and-the-cell-cycle/3962112d
- 18. Mutation in p53gene http://bio1151.nicerweb.com/Locked/media/ch18/p53_tumor-suppressor.html
- 19.
- 20. DNA Repair • Restorethe premutational damage to the normal base sequence • Failure in these systems can lead to a higher mutation rate • Some repair systems are very efficient at resorting the original sequence. • Others convert the original sequence into a permanent mutation.
- 21. DNA Repair (Cont’d) •Repair the damaged DNA base • Delete the damaged DNA and using the complementary sequences to restore the normal sequence Direct reversal Excision repair pathways Repair of double strand breaks
- 22. Direct reversal ofdamaged DNA • Photoreactivation by Photolyase enzyme.
- 23. Base Excision Repair(BER) • Repair of AP sites glycosylase AP endonuclease Excision exonuclease DNA polymerase DNA ligase Griffiths et al (2004)
- 24. Nucleotide Excision Repair (NER) Excinuclease •detects the distortions in double helix cause by the presence of abnormal base • Such distortions include pyrimidine dimer caused by UV light or aflatoxin induced mutation. Griffiths et al (2004)
- 25. Repair of double-strandbreaks • Double-strand break is a mutation in which both strands of the double helix were to break at sites that were close together. • If left unrepaired, this can cause a variety of chromosomal aberrations resulting in cell death or a precancerous state. • Double-strand breaks can arise spontaneously or induced by ionizing radiation. • Two mechanisms to repair these lesions are Nonhomologous end joining (NHEJ) and homologous recombination.
- 26. Nonhomologous end-joining (NHEJ) • Complementarystrand cannot exploited because both strands are damage. • No sister chromatid • Error prone mechanism • Imperfect repair • Better than leaving unrepaired Griffiths et al (2004)
- 27. Homologous Recombination • Utilize sisterchromatoid • Error free Griffiths et al (2004)
- 28. Human disease andDNA repair defect Disease Cancer susceptibility Symptoms Ataxia telangiectasia Lymphomas Telangiectases in skin and eyes, immune dysfunction Bloom syndrome Carcinomas, leukaemias, Lymphomas Facial telangiectases, photosensitivity Fanconi anemia Leukaemias pancytopenia Xeroderma pigmentosum Skin carcinomas, melanomas Skin and eye photosensitivity, keratoses http://www.ncbi.nlm.nih.gov/books/NBK21794/table/A2780/?report=objectonly
- 30. PTEN • Phosphatase andtensin homolog detected on chromosome Ten • Tumor suppressor protein • play a role in maintenance of tumor microenvironment, cellular senescence and cell cycle regulation • regulate DNA damage mechanism
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- 32. Objectives of thestudy • To investigate the role of phosphorylation • subsequent nuclear localization of PTEN with DNA repair. • Using PTEN deficient U87MG cells
- 33. Materials and Methods •MTT assay • Fluorescence microscopy • Western blotting and immunoprecipitation • Comet assay • Statistical analysis with Origin Pro v.8 software
- 34. Results from MTTassay Percentage of survivability is lowered in all transfected cells compared to untransfected cell
- 35. Results of Cometassay
- 37. Conclusion • DNA damageaugments phosphorylation of PTEN. • Nuclear accumulation of PTEN increases which positively regulates the DNA repair process. • PTEN as the intermediate component of DNA repair. • Phosphorylation of PTEN is associated with DNA repair • PTEN is essential for successful DNA repair.
- 38. References • Clancy S.DNA damage and repair: Mechanisms for Maintaining DNA Integrity. Nature Education. 2008;1(1):103. • Griffiths AJF, Wessler SR, Lewontin RC, Gelbart WM, Suzuki DT, Miller JH. Introduction to Genetic Analysis. 8th ed. W.H. Freeman; 2004. • Karp G. Cell and Molecular Biology: Concepts and Experiments. 7th ed. John Wiley and Sons Inc; 2013. • Wei, S., Shalhout, S., Ahn, Y.H. and Bhagwat, A.S. (2015). A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair. DNA repair 27(2015)9-18