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Download Ch. 14. Mutations and Repair
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sickle-cell anemia Analogy of the effects of substitution, deletion, and insertion of one letter in a sentence composed of three-letter words, demonstrating point and frameshift mutations. Standard base-pairing relationships (a), compared with anomalous base-pairing that occurs as a result of tautomeric shifts (b). The long triangle indicates the point at which the base bonds to the pentose sugar. NORMAL RARE FORM The standard basepairing relationships compared with two anomalous arrangements occurring as a result of tautomeric shifts. The long triangle indicates the point of bonding to deoxyribose Standard base-pairing relationships (a), compared with anomalous base-pairing that occurs as a result of tautomeric shifts (b). The long triangle indicates the point at which the base bonds to the pentose sugar. A : T to G : C transition mutation as a result of a tautomeric shift in adenine. A : T to G : C transition mutation as a result of a tautomeric shift in adenine. Tautomeric Shift in Adenine Formation of a T-double bond-A to a Ctriple bond-G transition mutation as a result of a tautomeric shift in adenine. Formation of a T-double bond-A to a Ctriple bond-G transition mutation as a result of a tautomeric shift in adenine. MUTAGENS: BASE ANALOGS 5-bromouracil (5-BU) in similar in structure to thymine. In the common keto form, 5-BU base-pairs normally with adenine, behaving as an analog of T. In the rare enol form, it pairs anomalously with guanine, behaving as an analog of C. In the common keto form, 5-BU basepairs normally with adenine, behaving as an analog of T. In the rare enol form, 5BU pairs anomalously with guanine, behaving as an analog of C. ALKYLATION [another way of possible chemical modifications of bases] Conversion of guanine (G) to 6-ethylguanine by the alkylating agent ethylmethane sulfonate (EMS): addition of ethyl on 6th carbon of the base. The 6-ethylguanine, being an analog of A. base-pairs with thymine MUTAGENIC RADIATION GOOD NEWS: MUTATIONS CAN BE REPAIRED Direct correction in replication: DNA polymerase proofreads in 3’ → 5’ direction • Synthesis of DNA strand has high rate of misincorporations (ERRORS), about 1 in 100,000 nucleotides! (10-5); with proofreading ~ 99% repaired; still 10-7 with • Mutations in gene that codes for the proofreading subunit → no proofreading!“mutator” gene of E. coli Mismatch repair (after proofreading, still during replication) AAACCTTGGG TTTGGATCCC • But how to decide which strand is correct? • In E. coli, DNA methylation of A on template (in GATC) strand happens during replication so template is in fact temporarily ‘highlighted’ by methylase enzyme • Repair enzyme recognizes mismatch, makes a ‘nick’; exonuclease degrades a portion; Dna pol + ligase fill the gap Post-replication repair occurs if DNA replication has skipped over a lesion, such as a thymine dimer (produced by UV). Through the process of recombination, the correct complementary sequence is recruited from the parental strand and inserted into the gap opposite the lesion. The new gap that is created is filled by DNA polymerase and DNA ligase. DNA ligase working on repair Damaged DNA repaired by photoreactivation repair. The bond creating the thymine dimer is cleaved by the photoreactivation enzyme (PRE), which must be activated by blue light in the visible spectrum. Base-excision repair (BER) accomplished by uracil DNA glycosylase, AP endonuclease, DNA polymerase, and DNA ligase. Uracil is recognized as a non-complementary base, excised, and replaced with the complementary base (C). Nucleotide-excision repair (NER) of a UV-induced thymine dimer. In actuality, 13 nucleotides are excised in prokaryotes and 28 nucleotides are excised in eukaryotes during repair. Two individuals with xeroderma pigmentosum. The 4-year-old boy on the left shows marked skin lesions induced by sunlight. Mottled redness (erythema) and irregular pigment changes in response to cellular injury are apparent. Two nodular cancers are present on his nose. The 18-year-old girl on the right has been carefully protected from sunlight since her diagnosis of xeroderma pigmentosum in infancy. Several cancers have been removed and she has worked as a successful model. Xeroderma pigmentosum, or XP, is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient. This disorder leads to multiple basaliomas and other skin malignancies at a young age. In severe cases, it is necessary to avoid sunlight completely. The most common defect in xeroderma pigmentosum is a genetic defect whereby nucleotide excision repair (NER) enzymes are mutated, leading to a reduction in or elimination of NER.[1] Unrepaired damage can lead to mutations, altering the information of the DNA in individual cells. If mutations affect important genes, like tumor suppressor genes (e.g. p53) or proto oncogenes then this disorder may lead to cancer. Patients exhibit elevated risk of developing cancer. Normally, damage to DNA in epidermal cells occurs during exposure to UV light. The absorption of the high energy light leads to the formation of pyrimidine dimers, namely CPD's (cyclobutane-pyrimidine-dimers) and 64PP's (pyrimidine-6-4-pyrimidone photoproducts). The normal repair process entails nucleotide excision. The damage is excised by endonucleases, then the gap is filled by a DNA polymerase and "sealed" by a ligase. Onset of skin cancers in patients with or without xeroderma pigmentosum (XP), The Dark Side of the Sun is a 1988 American - Yugoslavian drama film starring a young Brad Pitt about a young man in search for a cure for a dreaded skin disease. It is directed by Bozidar Nikolic. The footage for the film was shot in 1988 but due to the outbreak of civil war it had to be abandoned and much footage was lost. It was eventually released officially in 1997. An insertion sequence (IS), shown in red. The terminal sequences are perfect inverted repeats of one another. Site-directed mutagenesis. A single strand of DNA is isolated and hybridized with a synthetic oligonucleotide containing a triplet altered to encode an amino acid of choice. After semiconservative replication, a different complementary base pair is present in one of the new duplexes. Upon transcription and translation, a mutant protein that was “designed” in the laboratory will be produced. Structural organization of a copia transposable element in Drosophila melanogaster, showing the terminal repeats.