Molecular mechanism of DNA,damage n repair.pptx

Medical

1. Molecular mechanism of DNA and Chromosomal damage and repair

2. Molecular mechanism of DNA All organisms duplicate their DNA before each cell division.DNA replication is semi conservative because each strand in DNA double helix act as a template for new complementary strand and it is semi discontinous because when one strand is synthesized continously , the other strand is synthesized discontinously by Okazaki fragments.DNA replication is also bidirectional.

3. DNA replication One of the key molecules in DNA replication is DNA polymerase. They synthesize DNA and add nucleotide to the DNA chain including those that are complementary to the template. Replication occurs in a site called origin of replication . Proteins recognise the site ,bind and open up the DNA. As the DNA opens two Y shaped structures called replication forks are formed which forms replication bubble and moves in opposite direction as replication proceeds.Single stranded binding proteins coat, seperated strands of DNA, near replication fork,keeping them from coming back together into double helix.

4. DNA Polymerase add the first nucleotide at a new replication fork with the help of an enzyme called primase . Primase makes an RNA primer. A typical primer is about five to ten nucleotides long. The primer primes DNA synthesis, i.e., gets it started.Once the RNA primer is in place, DNA polymerase "extends" it, adding nucleotides one by one to make a new DNA strand that's complementary to the template strand .

5. A DNA double helix is always anti-parallel; in other words, one strand runs in the 5' to 3' direction, while the other runs in the 3' to 5' direction. This strand is made continuously, because the DNA polymerase is moving in the same direction as the replication fork. This continuously synthesized strand is called the leading strand. The other new strand, which runs 5' to 3' away from the fork , is made in fragments because, as the fork moves forward, the DNA polymerase (which is moving away from the fork) must come off and reattach on the newly exposed DNA. This strand, which is made in fragments, is called the lagging strand.

6. DNA Replication

7. The small fragments in lagging strand are called Okazaki fragments. The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The sliding clamp protien is a ring-shaped protein and keeps the DNA polymerase of the lagging strand from floating off when it re-starts at a new Okazaki fragment . Topoisomerase enzyme also plays an important maintenance role during DNA replication. It prevents the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opened up. The RNA primers are removed and replaced by DNA through the activity of DNA polymerase I , the other polymerase involved in replication. The nicks that remain after the primers are replaced get sealed by the enzyme DNA ligase .

8. Summary

9. Chromosomal damage and repair DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents that trigger DNA damage checkpoints have been applied to halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy.

10. Types of DNA Damage Oxidation, chemotherapy and radiation therapy can all damage DNA. There are many ways in which this can occur. Base Damage : A DNA base is chemically altered. This may cause a point mutation, or it may predispose to additional DNA damage. Base Mismatch : A mistake during DNA replication leads to insertion of the wrong base.This will cause a point mutation if it is not repaired. Pyrimidine Dimers : Two adjacent pyrimidine bases are cross-linked by ultraviolet light . This will cause a point mutation if it is not repaired prior to replication. • Intercalation : This occurs when abnormal chemical groups (such as chemotherapy drugs) are interposed in the DNA helix . This may prevent gene function and replication.

11. Crosslinking : This occurs when abnormal chemical bonds are formed within the DNA molecule. This may prevent gene function and replication, or cause DNA strand breaks. Single strand breaks (SSBs ): The sugar backbone is broken on one strand but not the other. • This is easily repaired as long as the other strand is still intact. Double strand breaks (DSBs): When the DNA is broken on both strands, it has "sticky ends" that can react with other DNA strands. This causes chromatid and chromosome aberrations that may be mutagenic or lethal. 

12. Measuring DNA Damage Peripheral Blood Lymphocyte Assay Peripheral blood lymphocytes are very sensitive to radiation, and you can count DNA aberrations in the blood: Conventional light microscopy generally measures unstable aberrations that disappear over days- months. Fluorescence in situ hybridization (FISH) can measure unstable and stable aberrations. Stable aberrations may persist for years. Total-body radiation doses >0.2 Gy will produce measurable chromosome aberrations in lymphocytes. A linear-quadratic mathematical model can be used to estimate absorbed dose from the number of aberrations.

13. DNA Repair DNA repair is a collection of process by which a cell identifies and corrects damage to the DNA molecules that encode its genome. The DNA repair processes is constantly active as it responds to damage in the DNA structure. Most cells posses three different categories of DNA repair systems: i ) Direct repair i ) Excision repair ii) Mismatch repair  

14. Direct repair Direct repair systems act directly on damaged nucleotides, converting each one back to its original structure. One common type of UV radiation mediated damages, pyrimidine dimers , are repaired by a light-dependent direct system called photoreactivation . When stimulated by light with a wavelength between 300 and 500 nm, the enzyme binds to pyrimidine dimers and converts them back to the original monomeric nucleotides

15. Excision repair Excision repair involves the excision of a segment of the polynucleotide containing a damaged site, followed by resynthesis of the correct nucleotide sequence by a DNA polymerase. These pathways fall into two categories: 1 . Base -excision repair Base excision repair involves removal of a damaged nucleotide base, excision of a short piece of the polynucleotide and resynthesis with a DNA polymerase. It is used to repair many minor damage like alkylation and deamination resulting from exposure to mutagenic agents. Enzyme DNA glycosylase initiates the repair process .

16. A DNA glycosylase does not cleave phosphodiester bonds, instead cleave the N- glycosidic bonds, liberating the altered base and generating apurinic or an apyrimidinic site, both called as AP sites. The resulting AP site is then repaired by an AP endonuclease repair pathway. These enzymes introduce chain breaks by cleaving the phosphodiester bonds at AP sites. This bond cleavage initiates an excision-repair process with the help of three enzymes_ Exonuclease DNA polymerase I DNA ligase

17. Nucleotide excision repair This is similar to base-excision repair, but is not preceded by the removal of a damaged base and can act on more substantially damaged areas of DNA. This repair system includes the breaking of a phosphodiester bond on either side of the lesion, on the same strand, resulting in the excision of an oligonucleotide . The excision leaves a gap that is filled by repair systems, and a ligase seals the breaks.

18. Mismatch repair The mismatch repair (MMR) system can detect mismatches that occur in DNA replication. Enzyme systems involves in mismatch repair are as follows: i ) Recognize mismatched base pairs. ii) Determine which base in the mismatch is the incorrect one. iii) Excise the incorrect base and carry out repair synthesis. A mismatched base pair causes a distortion in the geometry of the double helix that can be recognized by a repair enzyme system. it is important that the repair system distinguish the newly synthesized strand, which contains the incorrect nucleotide, from the parental strand, which contains the correct nucleotide . 

19. CROSSLINK REPAIR Radiation damage produces DNA-DNA and DNA- protein crosslinks Repair mechanism of these crosslinks is still under investigation Probably combination of Nucleotide Excision Repair and Homologous Recombinational Repair are needed for the repair. • Individuals with fanconi's anemia are hypersensitive to crosslinking agents.

20. REPAIR OF Single stranded DNA binding proteins Base excision repair Nucleotide excision repair . REPAIR OF Double stranded DNA binding proteins Homologous recombination repair Nonhomologous end joining Crosslink repair Mismatch repair

21. CHROMOSOMAL ABERRATIONS Aberrations seen at metaphase are of 2 types Chromosome aberrations Occurs early in interphase Before replication Chromatid aberrations Occurs in a single chromatid arm after chromosome replication and leaves the opposite arm of the same chromosome undamaged leads to chromatid aberrations.

22. EXAMPLES OF RADIATION INDUCED ABERRATIONS • LETHAL ABERRATIONS Rings ( dicentric & ring are chromosome aberrations) Anaphase bridge ( chromatid aberration) • NONLETHAL ABERRATIONS . Symmetric Translocation. e.g Burkitt lymphoma, leukemia. Deletion. e.g deletion of tumour suppressor genes leads to carcinogenesis

23. Conclusion Investigating DNA damage is essential in the diagnosis and prognosis of several cancers. The formation of some important biomarkers in the cells such as micronuclei (MN), nucleoplasmic bridges (NPB) and nuclear buds (NPB) can be used as an indicator of DNA damage due to exposure to cytotoxic or DNA damaging agents. Precision medicine is a new type of treatment that plays a critical role in selecting the most appropriate therapy at the suitable time, as it only succeeds in targeting the DNA repair pathway in cancer. This treatment strategy is based on a unique genetic background, environment and lifestyle for the individual

24. The innovative diagnostic methods including DNA damage analysis are improving gradually, especially for precision medicine, and help in analyzing a large amount of new potential biomarkers leading to facilitation of the detection of early disease stages and disease prognosis Finally, the application of precision medicine is the most progressively developed field which depends on the improvement in DNA damage analysis and the investigation of novel markers

25. DNA damage occurs on a daily basis by endogenous and exogenous sources. Distinct DNA repair systems recognize and remove the lesions. When the damage remains unrepaired DNA damage checkpoints can halt the cell cycle or induce cellular senescence or apoptosis. Erroneous repair or replicative bypass of lesions can result in mutations and chromosomal aberrations. When mutations affect tumor suppressor genes or oncogenes , cell might transform into cancer cells. Therefore, DNA repair is essential for preventing tumor development. However, once a cancer has developed, DNA damage can be exploited to reduce cancerous growth and evoke apoptotic demise of cancer cells. Thus, chemo- and radiotherapies are still today, over 60 years after having been first introduced into tumor therapy, important strategies to fight cancer.

26. Given the central role of genome instability in triggering and treating cancer, it is likely that genotoxic treatments will remain an important avenue of cancer therapy. Also the better understanding of DNA repair systems will allow therapies that specifically target selected repair pathways. It will be of particular importance to gain a deeper understanding how the various DNA repair systems interact with each other in the context of cellular homeostasis and DNA metabolism in order to optimize targeted approaches to cancer therapy.

27. Source – Radiobiology for the radiologist Eric J Hall Amato J Giacca

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