Cell cycle & DNA repair

Different phases of cell cycle along with diagram and DNA repair mechanism and DNA damage.

1. Cell Cycle & DNA repair M. Amil Rahman(SR) Department of Biochemistry

2. Introduction Definition Reproduction & division of cell feature of all living things. Cell cycle or cell-division cycle,  is the series of events that take place in a cell as a controlled set of events, which leads to cell growth and division. Prokaryotic Cell (Bacteria—without nucleus) cell cycle occurs as ‘ Binary Fission ’ Eukaryotic cell (with nucleus, human, animal & plant cells) cell cycle has 2 PRINCIPAL PERIODS INTERPHASE MITOTIC PHASE

3. Cell division is an unique & essential process by which fertilized egg forms mature organism Also hair, skin, blood, intestinal cells get renewed. Before dividing into 2 new cells the cell must copy its genetic information so that the new cell can get everything they need‖— Cytoplasmic organelles, membranes, structural proteins, RNA all are replicated continuously in cell cycle --- doubling of cell size

4. Phases of cell cycle • 4 distinct phases G1 Phase (starting phase or Growth phase) Gap between Mitosis of preceding round of cell cycle & DNA synthesis of current cycle –cells increase in Size, Growth phase for RNA—each Chromosome with SINGLE molecule DNA S Phase DNA synthesis & replication, amount of DNA doubled G2 Phase Gap between the DNA synthesis & decision to divide—check point control before mitosis (Spindle in formed M phase Cell division (Mitosis) & formation 2Diploid cells (Cytokinesis) G1+S+G2 Phases = INTERPHASE(12 to 24 Hours in a mammalian cell) M Phase 1 to 2 hours Phases of cell cycle

5. G0 Phase G0 Phase-- Post-mitotic quiescent and senescent stage[non-proliferative cells like Neuron , Skeletal muscle and myocardium ] If DNA damage (Cancer)  cellular senescence cell degradation. Semi-permanent G0 phase—kidney or liver disease. Labile cells like Skin, Gastrointestinal and Hematopoietic cells. Condensed, duplicated chromosome chromatid telomere centromere telomere

6. Check-Points G1 Phase ―Not Responsive to Mitogenic Stimuli . Biosynthesis process--- use of 20 amino acids to form millions of proteins (to be utilized during S-phase) Controlled by p53 gene---any resultant damage from previous cell cycle has been repaired . R estrict entry of cells with damaged DNA into S phase. S Phase S ynthesis is completed quickly so that the base pairs sensitive to external factors such as drugs mutagens (nicotine) are not affected G2 Phase G2 checkpoint control mechanism ensures---everything is ready to enter the M (mitosis) phase and divides, all damage to DNA done in previous cycle repaired Two cell-cycle checkpoints control the order and timing of cell-cycle transitionsG1͢S and G2M

7. Chromosomes are in single copies, DUPLICATED only once per cycle, in S phase Duplication & division should be carried out with precision Important because ‗ Genetic information contained in DNA ‘ & ‗ Near-perfect transmission is important for evolution of species ‘ Hence a cell-cycle control system‘ regulates timing & coordination

8.   Paul Nurse won Nobel Prize in 2001 Discovery of Cyclin & & CDK Cyclin & CDK - -Protein kinases cause ‗Phosphorylation‘ of specific proteins in the cell required for transition to next stage Thus stimulate cell cycle progression (negatively controlled; CDK inhibitors—suppress cell cycle in DNA damage) 4 types Cyclin : A, B, D, E Example: MPF (maturation promoting factor) is Cyclin-CDK complex Chromosomal Condensation by phosphorylation of Histone initiates G2—M phase Regulation of Cell Cycle

9. Cyclin Kinases Function D CDK4, CDK6 Restriction points at G1---S phase E, A CDK2 DNA synthesis, early S phase B CDK1 Transition at G2---M phase

10. Extracellular Regulation of Cell Cycle • ―MITOGENS • Growth Factors, Hormones, Cell to Cell interactions • Binds to specific receptors on cell surface Phosphorylation of target proteins Cascade Phenomenon causes changes in gene expression

11. DNA Repair

12. DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome . In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage , resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis .

13. All cells possess machinery by which damage to DNA can be eliminated and the original form of the DNA double helix is restored. Causes of DNA Damage(Endogenous & Exogenous) DNA damage during DNA replication can occur through: • Misincorporation of deoxynucleotides during replication • By spontaneous deamination of bases during normal genetic functions • From x-radiation that cause “nicks” in the DNA • From UV irradiation that causes thymine dimer formation • From various chemicals that interact with DNA. The rapid repair of DNA damages are necessary since they may be lethal to the cell or cause mutations that may result in abnormal cell growth.

14. TYPES OF DNA DAMAGE: Here we discuss the various types of damage to DNA, including: Oxidative damage, Hydrolytic damage, DNA strand breaks , and others.

15. BY RADIATION Radiation acts by damaging DNA. When it hurts us, it damages DNA in healthy cells which are doing their job keeping us alive and well. Radiation can damage DNA either by scoring a direct hit, or by breaking-up water. The broken water is very reactive and can cause damage to DNA (or anything else it comes across). When we use it in medicine, e.g . in radiotherapy , it acts by damaging DNA in cancer cells.

16. BY HYDROLYSIS: The covalent structure of DNA is unstable in aqueous solution. It tends to hydrolyze to its monomeric components, and they themselves are subject to various hydrolytic reactions. A single base transformation within a DNA molecule may be sufficient to cause a mutation, or inactivate the DNA. Phosphodiester bond and N-glycosyl bond cleavage occurs due to hydrolysis. Hydrolysis involves two steps: Depurination Deamination

17. BY OXIDATION: Mutations caused by oxidative DNA damage may contribute to human disease. Oxidation of G generates oxoG , is highly mutagenic bcz it can be base-pair with A as well as with C . I f i t p a ir wi t h A du r ing re p l i c a t i on give r ise G:C t o T :A transversion cause human cancer i.e. by free radicals.

18. Mechanisms of Repair Several methods are available. They are: A. Excision repair B. Photo reactivation C. Recombinational repair D. Mismatch repair.

19. A. Excision repair 1. Repair of Thymine Dimers • A UV specific “ endonuclease ” makes a “nick” in the affected DNA strand, usually on the 5' end of the dimer, and the defective segment comes out. • The enzyme “DNA polymerase I” (DNA pol I) synthesizes new DNA strand in the 5' to 3' direction with the 3' end of the “ nicked ” strand acting as the “ primer ” and the intact complementary strand serving as the template. • 5' to 3'-exonuclease activity of DNA pol I then removes the damaged sequence. • Lastly “ DNA ligase ” enzyme seals the gap between the newly synthesized segment and the main chain.

20. 2. Spontaneous Deamination of Cytosine to Uracil • The deamination of cytosine to form uracil can take place due to inherent instability of cytosine. On replication in such an event, an ‘A’ will be inserted to pair with the ‘U’ formed thus forming a mutation.

21. B. Photo reactivation Also called light induced repair . The enzyme photo reactivating (PR) enzyme brings about an enzymatic cleavage of thymine dimers activated by the visible light (300-600 m µ ) leading to a restoration of the monomeric condition. Characteristics of the Enzyme • Enzyme is found in all organisms. • Binds to “NN” dimers, including cytosine dimers, in the DNA. • Activated by visible light energy and brings about cleavage of “C-C” bonds of the dimer.

22. C. Recombinational Repair Also called as sister-strand exchange. In this process, the unmutated single stranded segment from homologous DNA is excised from the “good” strand and inserted into the “gap” opposite the dimer. • It occurs after the first round of DNA replication.

23. D. Mismatch Repair of DNA • Mismatch repair corrects errors made when DNA is copied. Mechanism: This mechanism corrects a single mismatch base pair, e.g. C to A rather than T to A or a short region of unpaired DNA. • The defective region is recognized by an endonuclease that makes a single-strand cut at an adjacent methylated GATC sequence. The DNA strand is removed through the mutation, replaced and relegated.

24. CLINICAL ASPECT 1. Xeroderma Pigmentosum • Transmitted as autosomal recessive • Genetic defect: DNA repair mechanisms are defective. DNA damage produced by UV irradiation specially thymine dimers, cannot be incised. Results from inborn deficiency of the enzyme “nicking endonuclease”. Clinical Manifestations • Increased cutaneous sensitivity to UV rays of sunlight. • Produces blisters on the skin. • Dry keratosis, hyperpigmentation and atrophy of skin. • May produce corneal ulcers. Prognosis: Fatal, death takes place due to formation of squamous cell carcinoma of skin.

25. 2.Ataxia telangiectasia • A familial disorder • Inheritance: Autosomal recessive • Increased sensitivity to X-rays and UV rays is seen. Clinical manifestations • Progressive cerebellar ataxia. • Oculocutaneous telangiectasia. • Frequent sinopulmonary infections. • Lymphoreticular neoplasms are common in this condition. • IgE deficiency has been demonstrated in 67 per cent of cases.

26. 3. Bloom’s Syndrome Chromosomal breaks and rearrangements are seen in this condition. • Genetic defect: Probably defective DNA-ligase. • Clinical Manifestations – Facial erythema – Photosensitivity – Telangiectasia.

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