Mechanisms of Mutation And DNA repair

Thet Su Win

MTMT/D-5736940

DNA and its mutation

• 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

Mutagenesis

http://www.usfca.edu/fac-staff/dever/mutation_andrepair.pdf

Spontaneous Mutations

• Arise from 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

Depurination

• Loss of a purine base, occurs spontaneously

• A mammalian cell loses about 10,000 purines in 20hrs cell generation period

Depurination produces an Apurinic site

http://www.nature.com/scitable/content/depurination-produces-an-apurinic-site-55400

Deamination

Deamination of (a) Cytosine and (b) 5-methylcytosine Griffiths et al (2004)

(5-methyluracil)

Errors in DNA replication

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.

Base insertion and deletion

Griffiths et al (2004)

Deletion

Insertion

Indel mutation

Induced Mutations

• Arise after 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)

Induced Mutations (Cont’d)

http://www.powershow.com/view/3d29b5-Yjg5N/Chapter_7a_-_DNA_mutation_and_repair_Mutation

Induced Mutations (Cont’d) • Radiation

– UV (nonionizing radiation)

– Ionizing radiation

• Other mutagens

– aflatoxin B1 (AFB1)

Classification of Mutations

Mutations

Small-scale mutations

(microalteration)

Point Mutations

Transition and Transversion

Silent mutation

Missense mutation

Nonsense mutation

Insertions

Deletions

Large-scale mutations

(macroalteration)

Amplification or duplication

Deletions

Inversions

Rearrangement

Mutation and genetic disorders

Progeria

• Autosomal recessive disorders

• rapid aging disease

• caused by single point mutation (in LMNA gene)

• Thin, bald, old-looking skin

Mutation and haematological disorders

• Sickle cell anaemia

• a result of single nucleotide polymorphism (SNP)

• Hb S

http://evolution.berkeley.edu/evolibrary/article/mutations_06

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

Mutated p53 and cancer

https://voer.edu.vn/m/cancer-and-the-cell-cycle/3962112d

Mutation in p53 gene

http://bio1151.nicerweb.com/Locked/media/ch18/p53_tumor-suppressor.html

DNA Repair mechanisms

DNA Repair

• Restore the 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.

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

Direct reversal of damaged DNA

• Photoreactivation by Photolyase enzyme.

Base Excision Repair (BER)

• Repair of AP sites

glycosylase

AP endonuclease

Excision exonuclease

DNA polymerase

DNA ligase

Griffiths et al (2004)

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)

Repair of double-strand breaks

• 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.

Nonhomologous end-joining (NHEJ)

• Complementary strand cannot exploited because both strands are damage.

• No sister chromatid

• Error prone mechanism

• Imperfect repair

• Better than leaving unrepaired

Griffiths et al (2004)

Homologous Recombination

• Utilize sister chromatoid

• Error free

Griffiths et al (2004)

Human disease and DNA 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

PTEN

• Phosphatase and tensin 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

http://jcs.biologists.org/content/114/13/2375/F2.expansion.html

Objectives of the study

• To investigate the role of phosphorylation

• subsequent nuclear localization of PTEN with DNA repair.

• Using PTEN deficient U87MG cells

Materials and Methods

• MTT assay

• Fluorescence microscopy

• Western blotting and immunoprecipitation

• Comet assay

• Statistical analysis with Origin Pro v.8 software

Results from MTT assay

Percentage of survivability is lowered in all transfected cells compared to untransfected cell

Results of Comet assay

Conclusion

• DNA damage augments 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.

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