DNA is a hair like, fiber like long structure and it is thinner. DNA is double stranded made by nitrogen bases. Watson and Crick proposed that DNA is 3 dimensional double helical structures. The DNA molecule is made up of two identical halves that run parallel through out the length of the molecule. The double stranded DNA is more stable than single strand DNA. The hydrogen bonds, hydrophobic interaction, electrostatic/ ionic interactions and sugar- phosphate bonds make strong stability in double stranded DNA. The DNA having different physical and chemical properties they are acid - base property, viscosity, sedimentation behavior, absorption, optical rotation and topology. Denaturation of DNA occurs when available of unfavorable conditions. In this denaturation process the two strands of native DNA gets separated from each other. The main factors that affecting to denaturate the DNA are higher temperatures, alkaline pH and lowering salt concentration. If DNA having favorable conditions after the denaturation process, the formation of native double helical DNA from the two complementory strands is called renaturation. This renaturation process is also called annealing of DNA.
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Some time the DNA gets damage in process of replication. The sudden heritable changes that occur with in the genetic material of an organism are called as mutations. The range of mutations is from gene to genome. The smallest unit of mutation is called muton. The agents that bring about mutations are called as mutagens. In prokaryotes there are two methods of detecting mutations they are replica plating technique derived by Lederberg et. al. and ames test derived by prince ames at. al. In eukaryotes there are different techniques are detecting mutations they are clB technique - this technique is derived by H. J. Muller, attached X- ray technique, Russel's technique - this technique is derived by Dr. Russel. The DNA gets damage through various methods. The direct DNA damage occurs when the DNA absorbs the UV - B photon. The UVB light damages thymine base pairs in DNA sequence. A disorder in one strand which reproductive enzymes that never copy. It produces tan and it activates the production of melanin. There are other names for the direct DNA damage are thymine dimmers, pyrimidine dimmers, cyclobutane pyrimidine dimmers (CPD's) and UV - endonuclease - sensitive - sites (ESS). The indirect DNA damage occur when human skin absorbes the UV- photon. The chromophore of skin absorbs the UV- photons but it does not have capacity to exchange the energy to harmless heat very fast. The molecules that they do not have capacity of long lived excited state. This lengthy life time guides to a high possibility for reactions with additional molecules - so called bimolecular reactions. Melanin and DNA are containing extremely tiny excited condition life time in the range of a few femtoseconds (10-15s). The excited condition of these molecules is 1,000 to 1,000,000 times higher than the life time of melanin. And for this reason they may affect damage to living cells or organisms which approach into contact with them.
5. METHODS and METERIALS:
Any damage that occurs within the DNA is usually repaired by DNA repair systems restoring to normal functions. If DNA damage escapes the repair system then it can be observed as mutation.
There are nearly five types of DNA repair system. They are
photo reactivation repair system
The presence of Photo reactivation repair was first discovered by Albert Kelnar in 1949 in E.coli. It is involved in the repair of pyramidine dimers produced due to U V exposure. It is direct repair system that can directly cleave the dimer bond. This repair system is a light dependent repair system that gets activated only in pressure of visible light. It is enzyme mediated. The enzyme that recognizes the pyramidine dimers is called as "Photo reactivation enzyme" or "photolyase". The enzyme can also bind to the pyramidine dimer even in the absence of light but requires energy to break the dimer bond.
The enzyme gets activated by utilizing a photon of visible light specifically in blue region of spectrum and the energy derived is used to break the dimer bond directly restoring normal function. any mutation within the gene PHR coding for the enzyme is not lethal to an organism indicating the presence of other repair system is generally absent in mammals including humans.
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5.2. EXCISSION REPAIR:
this is the most common technique of repair system of both prokaryotes and eukaryotes. it can repair any sort of damage to DNA both minor alterations or major alterations. This system involves three steps.
i) Incision step: an endonuclease activity of the repair system, recognizes the damage and induce nicks.
ii) Excision step: an endonuclease activity of the repair system removes the DNA carrying the damage producing a gap.
iii) Synthesis step: DNA polymerase I fills the gap by using other strand as template and DNA ligase covalently seals the final nick
the excission repair system is mainly separated into two types.
I. Base excision repair: it is involved in repair of minor chemical alterations that occur within the nitrogen bases induced by mutagens. They involve a group of enzymes called DNA glycolyases that can break the glycoside bonds that is connecting base together molecule which is called Apyramidine site (AP site). Each DNA glycolyase can recognizes a specifically chemical altered base. The enzyme breaks the glycosidase bond releasing Uracil generating Apyramidine site (AP site). AP site in the DNA are recognized by 'AP endonucleases'. That initiate nick in the DNA. the gap is then filled up by the DNA polymerase I binding to 3' end it adds nucleotide and nick is sealed by DNA ligase.
II. Nucleotide excision repair: this repair system involved in repair of major damage like structural distortions. This repair involves a specific excision nuclease activity called 'excinulease' that can initiate a nick on either side of damage and remove oligonucleotide sequence causing the damage. A trimeric protein made up of two subunits of UV r A and one subunit of UV r B recognises and binds to structural distortion in the DNA. UV r A2 is then released and UV rC then join UVrB- complex. in the UVr- BC complex, UVrB cleaves the phospho diester bond at the 3' end of the damage and UVrC cleaves the 8th phospho diester bond at the 5'end of the damage. UV rD which passes DNA helicase II activity unwinds the DNA releasing af 13 nucleotide sequence generating a gap in one strand that faces af normal strand. The gap is then filled up by DNA polymerase I by using other strand as template. DNA ligase seals the final nick. excinuclease activity in humans corresponding to the action of 17 polypeptides and removes an oligfonucleotide sequence of 29 nucleotides.
Disorder caused due to defect in genes coding for excision repair is 'Xeroderma pigmentose'. This leads to skin abnormalities and also 'skin cancer'. They are lightly sensitive to several lights. Conckayne syndrome is another disorder which is characterized by physical and neurological impairments.
5.3. RECOMBINATIONAL REPAIR:
It is a post replication repair system. This system does not remove the damage but minimizes or limits the damage to parental strand. This system involves a homologous recombination between the two daughter's duplexes involving single stranded exchange hence called recombinational repair. Due to replication of DNA molecules with structural distortion it produces two daughter duplexes of which one daughter duplex produced from damaged template carry damage in one strand facing a large gap in other strand while the other duplex produced from normal strand or normal template is normal. The gap in the first duplex is filled up by a homologous recombination between the two homologous strands of two daughter duplexes. as a result of recombination event, both the duplexes produced are abnormal. One duplex carries a distortion in one strand facing a normal newly synthesized strand and the other duplex carries a normal newly synthesized strand with a gap in potential strand. The gap in DNA duplex will be filled up by DNA polymerase I and DNA ligase seals the final nick. Hence at the end of recombinational repair the damage is limited to perantal strand only. recombinational repair should separates after every replication cycle until and unless the damage is repaired by excision repair.
5.4. SOS REPAIR SYSTEM:
This repair system acts as a final step when all the steps fail. This system activates only when the removal of the cell is in damage. This system minimizes the effect of the damage but do not remove completely. This system includes bypass system that allows the chain growth to proceed through damaged site by adding any nucleotides into the DNA, hence such type of repair system is also known as error prone repair system. The principal involved in this repair system is survival with mutations in better than certain death. Survival genes products are involved some are Lex A, Rec A, UV mutable (umuC) and umuD. Here Lex A keeps the sos repair in repressed condition. Rec A protein performs three important functions they are Rec A protein level increases and cleaves Lex A proteins, Rec A also binds to single strand DNA and this protein inhibits the editing function of DNA polymerase along with umuC and umuD. After 4 hours damage to the DNA, Rec A level decreases and Lex A level increases, there by the SOS repair goes back to repressed condition.
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5.5. MISMATCH REPAIR:
This repair system is involved in the repair of mismatches that are produced as replication errors. Replication through is an accurate process it is not 100% accurate. It can commit errors with frequency of 1 in 10 to the power of 9 N. these errors are then repaired by this repair system. This mismatch repair system operates at the end of every replication cycle to check the presence of mismatches. The most common type of mismatches that occurs is GT mismatches. GT base pair can be restored at either GC or AT. In order to restore to normal base pair the repair system should differentiate the newly synthesized strand from the parental strand. This is done by recognizing a specific sequence called GATC where adenine is methylated by both enzyme DAM methylase. The replication of fully methylated GATC sequence produces hemi methylated GATC sequence. In this hemimethylated condition, the repair system recognises the unmethylated strand as newly synthesised strand. Several gene products are involved in the mismatch repair system. Mut S recognizes and binds to mismatches present in the DNA. Mut H and Mut L they join the complex. Mut H has a GATC specific endo nuclease activity that can initiate a nick in the unmethylated strand either at 5' or 3' ends of a mismatch. Mut U has a DNA helicase II activity that unwinds the DNA. Different exonucleases are involved to remove a stretch of DNA based on the nick induced. If the nick is created at the 5' end of mismatch then exonuclease VII that passes 5' to 3' exonuclease activity is involved in E.coli. If nick is induced at 3' end of mismatch then exonulease I that passes 3' to 5' exonuclease activity is involved. DNA polymerase I fill up the gap using other strand as template while the final nick is covalently sealed by DNA ligase.