Notes on Molecular Biology of the Cell

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University of Toronto St. George
Kenneth Yip

DNA Damage Can Be Removed By More Than One Pathway There are two pathways that allow for the damage in DNA to be excised: 1. Base excision repair It involves a battery of enzymes called DNA glycolases, each of which recognize a specific altered base (e.g. deanimated Cs & depurinated As, oxidized bases, bases with open rings, C-C double bonds accidentally converted into C-C single bonds) in DNA and catalyze its hydrolytic removal In order to find an altered base, a key step is an enzyme-mediated flipping out of the altered base which allows DNA glycolase to assess the damage and evaluate each base. Once the enzyme finds a damaged base, it is removed from its sugar. The missing tooth created by DNA glycolase is recognized by the AP endonucleases which cuts the phosphodiester backbone, after which the damage is removed and the resulting gap is fixed Depurination also leaves a deoxyribose sugar with a missing base, which is repaired beginning with AP endonuclease, and then the rest of base exclusion repair. 2. Nucleotide Exclusion Repair This mechanism can repair the damage cause by any large change in the structure of DNA Such bulky lesions are created by the covalent reactions of DNA bases with large hydrocarbons, pyrimidine dimers (T-T, C-T) caused by sunlight. A large multi-enzyme complex scans the DNA for distortion, rather than a specific base. Once it finds a bulky lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away ss-oglionucleotide containing the lesion. The large gap produces is fixed by DNA polymerase and DNA ligase 3. Direct chemical reversal of DNA damage It is an alternative to base and nucleotide excision-repair process that is employed for the rapid removal of highly mutagenic or cytotoxic lesions Coupling DNA Repair to Transcription Ensures That Cells Most Important DNA Is Efficiently Repaired Cells have a way of direction DNA repair to the DNA sequences that most need it by linking RNA polymerase to the repair of DNA damage Transcription-coupled repair (TCR) o RNA polymerase stalls at DNA lesions and through the use of coupling proteins, directs the repair machinery to these sites o In bacteria, DNA is short so, the stalled RNA will dissociate from the DNA, and transcription restarts o In eukaryotes, a more complex reaction is used to back up RNA polymerase, repair the damage and then restart the polymerase o TCR is specific to the template strand of transcribe DNA; the other strand is repaired with the same speed and efficiency as DNA that is not transcribed at all o Its importance is seen in those with Cockaynes Syndrome, which is cause by a defect TCRRNA polymerase is permanently stalled at sites of DNA damage that lie in important genes The Chemistry of the DNA Bases Facilitates Damage Detection DNA helix is optimally constructed for repair, has a backup copy of all genetic information and the specific base-pairing of nucleotides allows easy detection of unnatural bases RNA is thought to have served as genetic material before DNA, so why did A, C, G, and U, later become, A, C, G, and T? o The spontaneous deamination of C yields uracil, which is relatively harmless, but is U was a natural base, then DNA glycolase would find it difficult to distinguish between a deanimated C or uracil In vertebrate DNA, selected C nucleotides are methylated at specific C-G sequences that are associated with inactive genes. The accidental deamination of methylated Cs yields thymine, which is now mismatched with a G on the opposite strand. To repair this, a special DNA glycolase recognizes the mismatched pair and removes the T. o Relatively ineffective because although only 3% of all Cs is methylated, the mutations at these sites lead to one third of all single- base mutations in inherited human diseases. Special DNA Polymerase Are Used in Emergencies to Repair DNA A different strategy is used when DNA damage is extensive, and is risky The highly accurate DNA polymerase stall when it encounters damaged DNA, and employs versatile, but less accurate back-up polymerases Humans have more than 10 such DNA polymerases, which recognize a specific type of damage, and specifically add the nucleotide needed to restore the original sequence The back-up polymerase are not as accurate, lack exonucleolytic proofreading, and are much less discriminating in choosing which nucleotide to initially incorporate, thus back-up polymerase are only allowed ass only one or few nucleotides Double-Strand Breaks Are Efficiently Repaired Ionizing radiation, replication errors, oxidizing agents, and other metabolites produced in cells can cause both strands of the DNA helix to breakvery dangerous o If left unrepaired, it can lead to the breakdown of chromosomes are the cell dividesloss of gene info There are two mechanisms that ameliorate this damage: 1. Nonhomologous end-joining is when broken ends are brought together and rejoined by DNA ligation, generally with the loss of one or more nucleotides at the site of joining This end-joining mechanism which is quick and dirty, is common in mammalian somatic cells Although a mutation occurs, since so little of the genome codes for protein this is an acceptable method to rejoin broken chromosomes By age 70, a typical human will have >2000 of these scarsthe specialized structures of telomeres prevents the natural ends of chromosomes from being mistaken for broken DNA 2. Homologous recombination is when the sister chromatid is used as a template to repair the defective strand to the original DNA sequence. Nonhomologous end-joining is predominate in humans, and the homologous recombination is only used during and shortly after DNA replication (S and G2 phases) DNA Damage Delays Progression of the Cell Cycle Cells delay the progression of the cell cycle until DNA repair is complete in order to maximized the effectiveness of their DNA repair enzymes The orderly progression of the cell cycle is maintained through the use of checkpoints that ensure the completion of one step before the next one can beginthe cell cycle stops at these checkpoints in damaged DNA is detected These delays facilitate DNA repair by providing the time needed for the repair to reach completion The importance of special signalling mechanisms that respond to DNA damage indicated by the phenotype of humans who are born with defects in the gene that encode the ATM proteinleads to the disease ataxia telangiectasia (AT) o The ATM protein is a large kinase needed to generate the intracellular signals that produce a response to many types of spontaneous DNA damage Chapter 6: How Cells read the Genome: From DNA to Protein When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the DNA is first copied into RNA transcription. It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein translation The flow of genetic information in cells is therefore from DNA to RNA to protein. All cells express their genetic information in this waya fundamental principle called the central dogma of molecular biology. Portions of DNA Sequence Are Transcribed into RNA Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. It differs from DNA chemically in two respects: 1. The nucleotides in RNA are ribonucleotidesthat is, they contain the sugar ribose rather than deoxyribose 2. Although, like DNA, RNA contains the bases a
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