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- 1. DNA REPAIR &MUTATIONS Ms. Najma Malik Demonstrator Biochemistry Department
- 2. DNA REPAIR Despiteproofreading and mismatch repair during replication, some mismatched bases do persist. In addition, DNA can be damaged by mutagens produced in cells or from the environment. The environmental mutagenic agents can be either radiations or chemicals. These mutagens damage DNA, causing mutations. If the damage is not repaired, a permanent mutation may be introduced that can result in any of a number do deleterious effects leading to cancer The maintenance of the integerity of the DNA is key of life. Consequently, all cells possess mechanisms to repair damaged DNA.
- 3. REPAIR MECHANISMS Themechanisms used for the repair of DNA involve First recognition of distorted region of the DNA Secondly removal or excision of the damaged region of the DNA strand Then filing of gap left by the excision of the damaged DNA by DNA polymerase. Finally sealing the nick in the strand that has undergone repair by the ligase.
- 5. Types of DNArepair system in E. coli System Enzymes/ Proteins involved in repair Type of damage Mismatch repair Dam methylase Mut H, Mut L, Mut S proteins DNA helicase II SSB DNA polymerase III Exonuclease I DNA ligase Mismatches Copying errors Base excision repair DNA glycosylases AP endonucleases DNA poymerase I DNA ligase Spontaneous, chemical or radiation damage to a single base. Pyrimidine dimers, alkylated bases. Nucleotide excision repair ABC excinuclease DNA polymerase I DNA ligase DNA lesions that cause large structural changes, e.g. pyrimidine dimers may be due to chemical, radiation or spontaneous. Direct repair DNA photolyases O6 -Methyguanine-DNA Methyltransferase Pyrimidine dimers, O6 - Methyguanine
- 6. The DNApolymerase has 3′ to 5′ exonuclease activity. Hence any mispaired nucleotide added is immediately removed TIC PROOFREADIN G
- 7. This takesplace along with the replication process (proof-reading). The original template DNA contains methylated residues (N6- methyl adenine and 5-methyl cytosine). The newly synthesized strand will not have methylated bases. So enzymes can recognize the original (correct) DNA strand. The mismatched base is identified and removed along with a few bases around that area. The wrong base is removed by the endonuclease activity. It removes 24 – 32 nucleotides around the wrong base. As the endonuclease cleaves at two points, the enzyme is sometimes also called excinuclease. A small segment of DNA with correct base sequence is then synthesized by DNA polymerase beta. Then the gap or nick is sealed by DNA ligase. NUCLEOTIDE EXCISION REPAIR
- 9. Specific proteinsscan the newly synthesized DNA strand (new strand is identified by not being methylated) (Fig. 44.18.). The mismatched area is identified, a loop is made. In E.coli, this recognition and looping is done by three proteins, MutS, MutC and MutH. Then that segment is removed. Finally correct segment is synthesized by the help of DNA polymerase, SSBs, and ligase. STRAND DIRECTED MISMATCH REPAIR
- 11. BASE EXCISION REPAIR Depurination of DNA is a spontaneous process, which occurs at a rate of 10,000 per cell per day. Thus cytosine, adenine and guanine bases spontaneously form uracil, hypoxanthine and xanthine respectively. These are not normal bases of DNA. Specific N- glycosylases can remove these abnormal bases. The sugar has no base. Apurinic endonucleases excise this abasic sugar. Proper base is then added by a repair DNAP and ligase.
- 12. TRANSCRIPTION COUPLED REPAIR The template strand of transcribed region responds immediately by stalling of RNAP at the site of damage. The enzyme backs away, repair proteins are recruited and the defect repaired by NER. Cockayne syndrome results from deficiency of proteins that help to recognize the halted RNAP.
- 13. DOUBLE STRAND BREAKREPAIR Doubles strand breaks are produces all the time by reactive oxygen species and ionizing radiationd. The “Ku” protein and protein kinases are involved in this mechanism.
- 15. MUTATIONS i. A mutationis defined as a change in nucleotide sequence of DNA. This may be either gross, so that large areas of chromosome are changed, or may be subtle with a change in one or a few nucleotides. ii. Mutation may be defined as an abrupt spontaneous origin of new character. iii. Statistically, out of every 106 cell divisions, one mutation takes place.
- 16. CLASSIFICATION OF MUTATIONS Mutations can result from substitution, deletion or addition of one or more nucleotides. Substitution of one nucleotide by another is the most common type of mutation. A point mutation is defined as a change in a single nucleotide. This change may have mis-sense or nonsense effects. Deletion or insertion of a single nucleotide leads to a frameshift effect.
- 17. 1. SUBSTITUTION Replacementof a purine by another purine (A to G or G to A) or pyrimidine by pyrimidine (T to C or C to T) is called transition mutation. If a purine is changed to a pyrimidine (e.g. A to C) or a pyrimidine to a purine (e.g. T to G), it is called a transversion. The point mutation present in DNA is transcribed and translated, so that the defective gene produces an abnormal protein.
- 18. 2. DELETION Deletions maybe sub-classified into i. Large gene deletions, e.g. alpha-thalassemia (entire gene) or hemophilia (partial) ii. Deletion of a codon, e.g. cystic fibrosis (one amino acid) 508th phenyl alanine is missing in the CFTR protein. iii. Deletion of a single base which will give rise to frameshift effect.
- 19. 3. INSERTION Insertions oradditions or expansions are sub- classified into: i. Single base additions, leading to frameshift effect. ii. Trinucleotide expansions. In Huntington’s chorea, CAG trinucleotides are repeated 30 to 300 times. This leads to a polyglutamine repeat in the protein. The severity of the disease is increased as the numbers of repeats are more. iii. Duplications. Gene duplication results from unequal crossing over of chromosomes during meiosis and plays an important role in evolution.
- 20. EFFECTS OF MUTATIONS A.SILENT MUTATION A point mutation may change the codon for one amino acid to a synonym for the same amino acid. Then the mutation is silent and has no effect on the phenotype. For example, CUA is mutated to CUC; both code for leucine, and so this mutation has no effect.
- 21. B. MIS-SENSE BUTACCEPTABLE MUTATION A change in amino acid may be produced in the protein; but with no functional consequences. For example, in the normal hemoglobin A molecule, the 67th amino acid in beta-chain (HbA b-67) is valine. The codon in mRNA is GUU. If a point mutation changes it to GCU, the amino acid becomes alanine; this is called Hb Sydney. This variant is functionally normal. A conserved mutation occurs when the altered amino acid has the same properties of the original one; e.g. glutamic acid to aspartic acid.
- 22. C. MIS-SENSE; PARTIALLYACCEPTABLE MUTATION In this type, the amino acid substitution affects the functional properties of the protein. HbS or sickle-cell hemoglobin is produced by a mutation of the beta-chain in which the 6th position is changed to valine, instead of the normal glutamate. Here, the normal codon GAG is changed to GUG (transversion). HbS has abnormal electrophoretic mobility and subnormal function, leading to sickle-cell anemia.
- 23. D. MIS-SENSE; UNACCEPTABLEMUTATION The single amino acid substitution alters the properties of the protein to such an extent that it becomes nonfunctional and the condition is incompatible with normal life. For example, HbM results from histidine-to- tyrosine substitution (CAU to UAU) of the distal histidine residue of alpha-chain. There is methemoglobinemia.
- 24. E. NONSENSE; TERMINATORCODON MUTATION A tyrosine (codon, UAC) may be mutated to a termination codon (UAA or UAG). This leads to premature termination of the protein, and so functional activity may be destroyed, e.g. beta-thalassemia. Or, a terminator codon is altered into a coding codon ( UAA to CAA). This results in elongation of the protein to produce run on polypeptide (Hb Constant spring)
- 25. F. FRAMESHIFT MUTATION This is due to addition or deletion of bases. From that point onwards, the reading frame shifts. A “garbled” (completely irrelevant) protein, with altered amino acid sequence is produced. An example, Normal mRNA AUG UCU UGC AAA...... Normal protein Met Ser Cys Lys....... DeletedU mRNA AUG CUU GCA AA......... Garbled protein Met Leu Ala ............. In this hypothetical example, deletion of one uracil changes all the triplet codons thereafter. Therefore, a useless protein is produced. Frame shift mutations can also lead to thalassemia, premature chain termination and run-on- polypeptide.
- 26. G. CONDITIONAL MUTATIONS Most of the spontaneous mutations are conditional; they are manifested only when circumstances are appropriate. Bacteria acquire resistance, if treated with antibiotics for a long time. This is explained by spontaneous conditional mutations. In the normal circumstances, wild bacilli will grow. In the medium containing antibiotic, the resistant bacilli are selected.
- 27. MUTAGENS AND MUTAGENESIS Any agent which will increase DNA damage or cell proliferation can cause increased rate of mutations also. Such substances are called mutagens. X-ray, gamma-ray, UV ray, acridine orange, etc. are well-known mutagens. Müller (Nobel Prize, 1946) showed that the rate of mutation was proportional to the dose of irradiation. Beadle (Nobel prize, 1958) showed that the effect of X- irradiation on metabolism was due to mutations of genes. Tatum (Nobel Prize, 1958) further showed that a mutation of a single gene resulted only in a single chemical reaction, which gave evidence to the concept of “one gene, one enzyme”.
- 28. MANIFESTATIONS OF MUTATIONS i.Lethal Mutations The alteration is incompatible with life of the cell or the organism. For example, a mutation which does not produce alpha chains (4 gene deletion) will result in intrauterine death of embryo. ii. Silent Mutations Alteration at an insignificant region of a protein may not have any metabolic effect.
- 29. iii. Beneficial Mutations Although rare, beneficial spontaneous mutations are the basis of evolution. Such beneficial mutants are artificially selected in agriculture. Normal maize is deficient in tryptophan. Tryptophan-rich maize varieties are now available for cultivation. Microorganisms often have antigenic mutation. These are beneficial to microorganisms (but of course, bad to human beings). iv. Carcinogenic Effect The mutation may not be lethal, but may alter the regulatory mechanisms. Such a mutation in a somatic cell may result in uncontrolled cell division leading to cancer. Any substance causing increased rate of mutation can also increase the probability of cancer. Thus all carcinogens are mutagens.
- 30. SITE-DIRECTED MUTAGENESIS MichaelSmith (Nobel prize, 1993) described this technique. An oligodeoxyribonucleotide is synthesized, whose sequence is complementary to a part of a known gene. A specific deletion/insertion is produced in the oligo. It is then extended by DNAP. After replication, one strand is normal and the other strand contains the mutation at the specific site. This allows study on the effect of that particular mutation.