ECDA August 2009

Genetic disorders is now known to be a result of alterations of DNA sequences which correspondingly results in alterations of the amino acid sequence of the protein product. The consequence then is an altered conformation and function of the protein

DNA sequence Amino acid sequence Protein conformation Protein function

DNA MUTATION

MUTATION  Any alteration in gene sequence constitute a mutation. Mutation involves a change in the shape, structure or nucleotide sequence of the DNA  Mutation may be spontaneous or induced by agents called mutagens  Mutation may be lethal to the organism or it may lead to the development of new species

MUTATION  Mutation in the gene may be small or submicroscopic affecting only one of a small number of nucleotides, hence are called point mutations  Mutation in the gene can be large and be seen under the light microscope in the case of gross chromosomal abnormalities

POINT MUTATIONS  A point mutation can be a change in a single base or addition or removal of one or more nucleotides in the DNA  Single base changes are of 2 types:  Transition – involves a change of a pyrimidine to another pyrimidine or a purine to another purine T Cor G A  Transversion – involves a change of a purine to a pyrimidine or a pyrimidine to a purine T AG T C GA C

POINT MUTATIONS  Point mutations may have no effect on the amino acid sequence of the protein only if the substituted base is the third in the codon.  Ex. CCA CCG proline proline  Point mutation may result in the incorporation of a different amino acid in the protein, called a missense mutation  Ex. CCACGA proline arginine

POINT MUTATIONS  The mutation may result in the premature appearance of a stop codon resulting in a shorter protein which is likely to be nonfunctional  Ex. AAG UAG Lysine Term  Ex. UGG UAG Tryptophan Term  Ex. UAU UAG Tyrosine Term

FRAMESHIFT MUTATIONS  Addition and removal of one or more nucleotides in DNA, called insertion and deletion, respectively, may result in frameshift mutations  Ex. For normal strand shown AUGCGGUCUUGCAAAGGC... met arg ser cys lys gly A mutation causing deletion of base uracyl AUGCGGCUUGCAAAGGC... met arg leu ala lys ala

FRAMESHIFT MUTATIONS  Ex. For normal strand shown AUGCGGUCUUGCAAAGGC... met arg ser cys lys gly A mutation causing insertion of base adenine after the base in position 6 AUGCGGAUCUUGCAAAGGC... met arg ile leu gln arg

PHYSICAL MUTATIONS  Physical agents include ultraviolet (UV) and ionizing radiations.  The base most commonly affected by UV radiation is thymine.  When hit by high energy photons, diradicals are formed and can pair up forming covalent bonds.  For thymine, the major product is the thymine- thymine cyclobutane dimer (T-T dimer)

T-T dimer

CHEMICAL MUTATIONS Mutations caused by: alkylating agents deaminating agents intercalating agents

CHEMICAL MUTATIONS  Alkylating agents  The largest class of “potential” mutagens present in the environment  N-nitrosoamines found in cigarette smoke is metabolized by liver enzymes to form alkylating agents  Guanine is the most reactive among the four bases toward nearly all alkylating agents  When guanine is alkylated, it may be ignored resulting in a deletion in the daughter strand

CHEMICAL MUTATIONS  Deaminating agents  Sodium nitrite is used as a preservative, color enhancer, and color fixative in bacon, smoked fish, tocino, etc.  When ingested, sodium nitrite is converted to nitrous acid in acidic conditions.  Nitrous acid is a deaminating agent and removes groups from adenine, guanine, and cytosine.  Deamination of adenine results to hypoxanthine, a structural analogue of guanine, hence may base pair with cytosine resulting in transition

CHEMICAL MUTATIONS  Intercalating agents  These substances bind to DNA by becoming inserted between adjacent base pairs because of their flat ring structures  Benzopyrene, found in automotive exhaust and cigarette smoke  Benzene, an organic solvent  Aflatoxin, a metabolic product of molds in peanuts, oils, and grains

VIRAL MUTAGENS  Some viruses contain oncogenes which can be activated once they insert their DNA in the host’s genome or DNA  The process of inserting these viral genes is called lysogeny.  When viral DNAs are inserted into the host’s DNA, the sequence of the bases of the host’s DNA may be altered resulting in altered protein product, or activation of certain destructive genes.  Oncogenes – cancer-causing genes/DNA

DNA REPAIR MECHANISMS

DNA REPAIR  Cells are equipped with enzymes that can repair changes in the structure of their DNA  Photolyase – a photoreactivating enzyme that recognizes pyrimidine dimers and monomerizes it upon absorption of visible light

DNA REPAIR  Cells produce antioxidants such as glutathione and metallothione, a low molecular weight protein  Vitamins such as Vitamin C, A, and E can function as free radical scavenger  By binding to free radical elements, the resulting product can be simply excreted from the organism

DNA REPAIR  EXCISION REPAIR  A UV-specific endonuclease defects dimers and makes a cut near the dimer  The segment containing the dimer peels away and DNA polymerase synthesizes DNA from the 5’ to the 3’ direction  DNA polymerase which has a 5’ to 3’ exonuclease activity cuts the segment containing the dimer  A DNA ligase joins the newly synthesized DNA to the original DNA

DNA REPAIR

INHIBITORS OF GENETIC MECHANISMS

 Infection is a major worldwide problem. Like any other organisms, infectious agents must undergo cell division and thus genetic mechanisms are important for their growth and survival.  In the S phase of Cell Division process, DNA synthesis and duplication occur. Thus, to prevent microbial proliferation, inhibition of genetic mechanisms must happen. This is possible with the use of antibiotics.

ANTIBIOTICS  STREPTOMYCIN  Cause cell death by binding to the small subunit (30s) of the prokaryotic ribosomes preventing binding of mRNA  anti-TB drug

ANTIBIOTICS  CHLORAMPHENICOL  Binds with the 50s subunit of the prokaryotic ribosomes blocking the action of peptidyl transferase and preventing the attachment of mRNA- 30s complex to this large subunit  Drug against typhoid fever

ANTIBIOTICS  ERYTHROMYCIN  Binds to the 50s subunit preventing translocation  Drug against gram (+) bacteria

ANTIBIOTICS  Tetracycline  Interacts primarily with the small subunit to prevent the binding with amino acyl tRNA  In DNA, it intercalates between DNA bases resulting in change of the conformation of the DNA

QUESTIONS