Cell cycle and DNA repair mechanisms; Mutation

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

2. G0 phase

3. G1 phase
 1st 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- 2nd round of DNA synthesis
occurs
 Each cell will undergo this process once
only during the life span

5. G2 phase
 2nd 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 G1 or G0 phase

7. G0 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

11. 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

12.  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

13. 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

15. DNA REPAIR

16.  Human being- 1014 nucleated cells
 Each cell- 7 109 bp DNA
 1016 cell divisions in life-time
 If 10-10 mutations per bp per cell
generation escape repair– one
mutation per 106 bp
 may be maximum are futile

19. DNA MISMATCH REPAIR

20. 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)

21. 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

22. BASE EXCISION REPAIR

23. 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

25. Xeroderma pigmentosum

26. 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 !!!

28. Multi-step mechanism of DNA DS break repair

29. 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

30. Diseases due to defective DNA repair
mechanisms

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

32. EK BREAK TOH BANTA HAI

33. APOPTOSIS
PROGRAMMED CELL DEATH

34.  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

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

36. Death inducing
signals
1. DNA damage
2. Oncogene -induced proliferation
3. Loss of attachment to extracellular
membrane
4. Radiotherapy and chemotherapy

37. 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

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

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

40. 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

41. MUTATION

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

43. 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

44. 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

45. 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

46. Mis-sense mutation
1. Acceptable– Hb-Hikari ; Asp replaces
Lys at 61st position of β-chain- normal
function unaltered
2. Partially acceptable – HbS (Sickle
cell disease) ; β6 Glu → Val
3. Unacceptable – HbM
(Methaemoglobin);
α 58 His → Tyr; Non-functional Hb

47. 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

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

49. www.vpacharya.com