Cell cycle, DNA repair and mutation

Cell cycle and DNA repair mechanisms; Mutation

1. Prof. Dr. V.P.Acharya Cell cycle &DNA repair

2. G phase

3. G 1 phase 1 st gap phase Preparative phase Genomic DNA is checked for integrity Enzymes necessary to replicate the genome are produced Building materials for mitosis begin to accumulate If DNA damage not repaired → apoptosis

4. S phase Synthetic phase DNA is replicated once and once only Prokaryotes- 2 nd round of DNA synthesis occurs Each cell will undergo this process once only during the life span

5. G 2 phase 2 nd gap phase Cell prepares for mitosis Tubulin for spindle is produced If DNA damage detected- repaired

6. M phase Mitotic phase After M phase cells may re-enter G 1 or G phase

7. G phase Resting phase Most of the cells enter this state At the hour of need again enter cell cycle Cancer cells fail to enter this phase

8. Cyclins & cyclin dependent kinases Cyclins - a gr of proteins that turn on & off in cell cycle CDK - Cyclins activate them - Phosphorylate different substrates Cyclins + CDK= complex- act on different substrates

9. Cyclin D activates CDK- 4 & 6 ↓ Cyclin D+ CDK- 4 & 6 assemble as a unit in late G1 phase ↓ Behave as active Ser- Thr protein kinase ↓ Phosphorylation of Rb protein occurs ↓ Release E2F release from Rb repression ↓ Histones and DNA replication proteins starts ↓ Replication continues

10. Oncogenes & Oncoviruses target these check-points B-cell lymphoma- bcl oncogene is the CyclinD1 gene Oncoproteins from DNA viruses target the E2F release by relieving repression by Rb- unchecked cell growth

11. Replication licensing DNA is synthesised once and once only Secret is out in past years dissociation and/or cyclin -CDK phosphorylation subsequent degradation of several origin binding proteins that play critical roles in replication complex formation

12. DNA REPAIR

13. Human being- 10 14 nucleated cells Each cell- 7  10 9 bp DNA 10 16 cell divisions in life-time If 10 -10 mutations per bp per cell generation escape repair– one mutation per 10 6 bp may be maximum are futile

14. DNA MISMATCH REPAIR

15. Mismatch repair Corrects errors when DNA is copied. Ex- C replaced by A Specific proteins scan the new DNA Template strand methylated by Dam methylase enzyme GATC sequence as the reference point A is methylated in the sequences GATC endonuclease cuts the segment Up to 1000 bp distant repair can be done Repair done according to the message coded in the template strand Mechanism not clear for mammals and higher eukaryotes Clinical significance- HNPCC (Hereditary nonpolyposis Colon Cancer)

16. Base excision repair C ,A, G bases in DNA spontaneously form U, hypoxanthine or xanthine (Thermal lability) respectively-- Depurination They’re identified by specific N- glycosylases ↓ Remove the base from DNA ↓ Apurinic- apyrimidinic endonuclease excise the abasic sugar ↓ Polymerase fills the gap ↓ Ligase nicks the strands

17. BASE EXCISION REPAIR

18. Nucleotide excision repair Damaged DNA of up to 30 bases can be repaired A stretch of DNA (24-32 bp) flanking the defect is removed Cause of damage – UV light- Pyrimidine dimer formation -- Smoking – benzo [a] pyrene -guanine adducts -- Chemotherapeutic agents -- Chemicals Exinuclease Disease associated- Xeroderma pigmentosum 9 proteins involved- XPA- XPG involved in recognition and excision

19. Xeroderma pigmentosum

20. Double strand break repair Causes of damage– Oxygen free radical generation Ionizing radiation Chemotherapeutic agents 2 proteins are involved in non-homologous rejoining of a ds break Ku & DNA- Pk - heterodimers Ku first binds to free ends of DNA DNA- Pk approximates both the ends Base pairing occurs Extra tails cut Gaps closed by DNA ligase Defects if detected in G0/ G1 phase this method If defect detected in S, G2 or M phase- Homologous recombination occurs !!!

21. Multi-step mechanism of DNA DS break repair

22. Other repair mechanisms 5. Direct repair- damage is rectified by reversal with DNA photolyases 6. Post- replication / Recombination repair – immediately after replication – exchange with sister strand

23. Diseases due to defective DNA repair mechanisms

24. Some repair enzymes are multifunctional!!! Repair Components of transcription complex Cell cycle regulation Gene rearrangements

25. EK BREAK TOH BANTA HAI

26. APOPTOSIS PROGRAMMED CELL DEATH

27. Balance between life and death of cells. Self-defense mechanism – destroying infected cells Destroying cells that harbor genetic alterations (mutated) maintaining the genetic consistency and preventing the development of cancer

28. Ultra structural features Nuclear condensation & fragmentation Segregation of cytoplasmic organelles into distinct regions Blebs of plasma membrane Step ladder pattern of DNA in electrophoresis Membrane- bound cellular fragments which often lack nuclei

29. Death inducing signals DNA damage Oncogene -induced proliferation Loss of attachment to extracellular membrane Radiotherapy and chemotherapy

30. p53 – The guardian of genome p53 binds to damaged DNA ↓ Arrests the cell in G1 phase ↓ Directs the DNA repair mechanisms to the site for repair ↓ If DNA can’t be repaired p53 activates apoptotic mechanisms

31. Most of the oncogenic viruses and carcinogens target p53 ↓ Bind and inactivate it ↓ Mutated DNA allowed to proliferate ↓ cancer

32. Pro-apoptotic genes C- fos Rb P53 Apoptosis protecting genes Bcl-2 Other Oncogenes Activation of caspases - leads to cell death

33. Caspases- Cysteinyl aspartate specific proteases Caspases 1-10 (in order of their discovery) Caspase-8 – The initiator Caspase-3 – The Yama – Final caspase Cyt C released from Mitochondria ↓ Activates caspase activation cascade ↓ DNA breaks and protein breaks

34. MUTATION

35. Permanent changes in DNA sequence Germ cell mutation – Inherited diseases Somatic cell mutation – Cancer or malfunctions

36. Types of mutation Base substitution or Point mutation (Transition or Transversion type) Frame shift mutation Silent Mis-sense Nonsense Deletion Insertion Acceptable Partially acceptable Unacceptable

37. Point mutation Only one base is altered Protein with abnormal AA sequence May be of 2 types : Transition – Purine replaced by purine or Pyrimidine replaced by Pyrimidine Transversion – Purine replaced by Pyrimidine or vice-versa

38. Effect of Point mutation Silent mutation – no change in AA sequence; Degeneracy responsible Mis-sense mutation – Different AA sequence ; can be acceptable, partially acceptable or unacceptable Nonsense mutation – A stop codon is inserted; non-functional protein e.g. Thalassaemia

39. Mis-sense mutation Acceptable – Hb- Hikari ; Asp replaces Lys at 61 st position of β -chain- normal function unaltered Partially acceptable – HbS (Sickle cell disease) ; β 6 Glu → Val Unacceptable – HbM (Methaemoglobin); α 58 His → Tyr; Non-functional Hb

40. Frame shift mutation Insertion or deletion of one or two nucleotides in DNA Whole reading frame alters Deletion frame shift mutation – Cystic fibrosis of pancreas Insertion frame shift mutation -- Thalassaemia

41. When GOD solves your problems, you have faith in HIS abilities; when GOD doesn't solve your problems HE has faith in your abilities.  

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