A Seminar PresentationA Seminar PresentationononDNA Damage and Repair DNA Damage and Repair Presented by,Samhita KalitaMSc. Biotechnology ( 1st Sem. )Centre for Studies in BiotechnologyDibrugarh University

DNA DAMAGE

Occurs due to environmental factors and normal metabolic processes inside the cell, at a rate of 1,000 to 1,000,000 molecular lesions per cell per day.

This constitutes only 0.000165% of the human genome’s approximately 3 billion base pairs.

Unrepaired lesions in critical genes (such as tumor suppressor genes ) can impede a cell’s ability to carry out its function and appreciably increase the likelyhood of tumor formation.

The vast majority of DNA damage affects the primary structure of the double helix ; i.e. the bases itself are chemically modified.

DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level.

DNA is supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

Sources of damage Subdivided into two main types:

endogenous damage attack by reactive oxygen species produced from normal

metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination; also includes replication errors.

exogenous damage

caused by external agents such as ◦ ultraviolet [UV 200-300nm] radiation from the sun◦ other radiation frequencies, including x-rays and gamma

rays

◦hydrolysis and thermal disruption◦human-made mutagenic chemicals, especially

aromatic compounds that act as DNA intercalating agents

◦cancer chemotherapy and radiotherapy

The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones.

Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable.

Types of damage Five main types of damage to DNA due to endogenous

cellular processes:

oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species

alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine

hydrolysis of bases, such as deamination, depurination etc.

"bulky adduct formation" (i.e. benzo[a]pyrene diol epoxide-dG adduct)

mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.

Damage caused by exogenous agents comes in many forms. Some examples are:

UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage.

UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.

Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands.

Thermal disruption at elevated temperature increases the rate of depurination and single strand breaks,

e.g. hydrolytic depurination is seen in the thermophilic bacteria, which grow in hot springs at 40-80 °C. The rate of depurination (300 purine residues per genome per generation) is too high in these species.

Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as polycyclic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and Crosslinking of DNA just to name a few.

DNA REPAIR DNA repair refers to a collection of processes by which a cell identifies

and corrects damage to the DNA molecules that encode its genome.

DNA repair system is a complex or collection of enzymes i.e. Repair enzymes.

There are around 5-7 repair systems having more than 50 repair enzymes.

Four different categories of DNA repair system:

A. Direct repair system B. Excision repair system C. Mismatch repair system D. Recombination system

DIRECT REPAIR SYSTEM

Act directly on damaged nucleotides, converting each one back to its original structure.

Direct repair systems fill in nicks and correct some types of nuclear modification.

Damaged nucleotide can be repaired directly:

Nicks can be repaired by a DNA ligase if all that has happened is that a phosphodiester bond has been broken, without damage to the 5-phosphate and 3-hydroxyl groups of the nucleotides either side of the nick ( Figure A ). This is often the case with nicks resulting from the effects of ionizing radiation.

Figure A. Repair of a nick by DNA Ligase

Cyclobutyl dimers are repaired by a light-dependent direct system called photoreactivation.

Photoreactivation is a widespread but not universal type of repair: it is known in many but not all bacteria and also in quite a few eukaryotes, including some vertebrates, but is absent in humans and other placental mammals.

In E. coli, the process involves the enzyme called DNA photolyase (deoxyribodipyrimidine photolyase).

When stimulated by light with a wavelength between 300 and 500 nm the enzyme binds to cyclobutyl dimers and converts them back to the original monomeric nucleotides.

EXCISION REPAIR SYSTEM

Excision repair pathways fall into two categories:

a. Base excision repair

involves the removal of the damaged base from its sugar linkage and replaced. The Glycolase enzymes are the one which cut the base-sugar bond.

Example : uracil glycosylase - enzyme which removes uracil from DNA.

This enzyme has the property of cleaving the glycosidic bond of a corresponding specific type of altered nucleotide, thereby leaving a deoxyribose residue in the backbone. This site is called as apurinic or apyrimidinic site (AP site).

The deoxyribose residue is then cleaved by AP endonuclease , thus it is removed.

DNA polymerase adds the correct nucleotide to fill the gap and finally DNA ligase seals the nick.

b. Nucleotide excision repair

This system works on DNA damage which is "bulky" and creates a block to DNA replication and transcription (UV-induced dimers).

It probably recognizes not a specific structure but a distortion in the double helix.

The mechanism consists of 3 basic steps: i. Incision step – damaged structure is recognised by an endonuclease that

cleaves the DNA strand on both sides of the damage (~ 12 bases apart on both sides).

ii. Excision step - 5´- 3´ exonuclease removes a stretch of the damaged DNA.

iii.Synthesis step - DNA polymerase enzyme synthesizes a new replacement strand using single stranded DNA as template. Finally, DNA ligase seals the nick.

MISMATCH REPAIR SYSTEM

Corrects errors of replication, by excising a stretch of single-stranded DNA containing the offending nucleotide and then repairing the resulting gap.

This system repairs the errors caused by DNA polymerase during replication.

The system includes atleast 12 protein components that functions in the repair process.

RECOMBINATION SYSTEM

This is used to mend double strand breaks.

This repair mechanism promotes recombination to fix the daughter strand gap.

Used primarily to repair broken DNA ends such as are caused by ionizing radiation and chemical mutagens with similar action is the non-homologous end-joining reaction.

The Ku70, Ku80, and DNA-dependent protein kinase proteins are needed for non-homologous end-joining.

DEFECTS IN DNA REPAIR UNDERLIE HUMAN DISEASES

The symptoms of xeroderma pigmentosum include hypersensitivity to UV radiation, patients suffering more mutations than normal on exposure to sunlight, which often leads to skin cancer ( Lehmann, 1995).

Trichothiodystrophy is also caused by defects in nucleotide excision repair, but this is a more complex disorder which, although not involving cancer, usually includes problems with both the skin and nervous system.

Ataxia telangiectasia is a human autosomal recessive hereditary disease which causes several defects including about a hundred-fold increase in cancer susceptibility. AT patients' cells in culture show abnormalities including spontaneous and radiation-induced chromosome breaks and sensitivity to killing by X-rays.

However, AT cultured cells do not show a defect in repair of X-ray damage to their DNA; instead, unlike normal cells, they continue to replicate their DNA even when it has been damaged by X-rays.

REFERENCES

T. A. Brown: Genomes 2, Second Edition, BIOS Scientific Publishers, Ltd.

Dr. Gunter Obe: Mutations in Man, DNA Repair, p 35 : 53 Zeeland AA van, Natarajan AT, Verdegaal- Immerzeel PAM,

Filon AR (1980) Photoreactivation of UV induced cell killing, Mol Gen Genet 180: 495

C Emmanuel, Rev. Fr. S Ignacimuthu s. j., S Vincent: Applied Genetics Recent Trands & Techniques, www.mjppublishers.com

E. C. Friedberg, "Xeroderma pigmentosum, Cockayne's syndrome, helicases, and DNA repair: what's the relationship?", Cell 71: 887-889, 1992.

"Molecule of the year: the DNA repair enzyme", D. E. Koshland, Science 266: 1925, 1994.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi http://research.chem.psu.edu/sjbgroup/projects/translesion.

htm

THANK YOU