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Dna Repair Pathways
- 1. Genetic stability of organisms Accurate DNA replication + DNA repair mechanisms Present in prokaryotes and eukaryotes
- 2. 1 million individual lesions/cell/day • Heat • Metabolic accident • Radiation • Environment • depurination But only 1/1000 accidental base change results in a permanent mutation Molecular Biology of the Cell
- 3. Danger of DNA damage • Structural damage -> prevent replication/transcription – Stalling of replication fork • Harmful mutations -> impair survival of organism – Mutation in tumour suppressor genes for examples • If unrepaired: – Senescence – Apoptosis – Aberrant cell division -> cancer
- 4. Genetic diseases associated with defects in DNA repair Transcription-coupled repair HNPCC ATM Breast, ovarian and Defect in homologous colon cancer recombination, BRCA2 2005 W. H. Freeman Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed.
- 5. the DNA molecule can be repaired fairly easily because it carries 2 separate copies of all the genetic information Only viruses, that have a tiny genome (therefore tiny target for DNA damage) can afford to encode their genetic information in any molecule other than double stranded DNA single-stranded DNA or RNA.
- 6. Multiple DNA repair pathways • Base excision repair (BER) • Nucleotide excision repair (NER) • Mismatch repair (MR) Level of damage • DNA strand cross link repair • Homologous recombination (HR) • Non-homologous end joining (NHEJ) + transcription coupled repair
- 7. After T. Lindahl, Nature 362:709–715, 1993 Spontaneous DNA alteration Nucleotides known to be modified by: • Oxidative damage • hydrolytic attack • uncontrolled methylation Purines (guanine and adenine) are more affected by those spontaneous reactions. 5000 purine bases are lost every day: DEPURINATION Spontaneous DEAMINATION of cytosine uracil occurs at a rate of 100 bases per cell per day. UV can covalently link two adjacent pyrimidine bases to form THYMINE DIMERS.
- 8. substitution deletion
- 9. Base excision repair Nucleotide excision repair
- 10. “Risky Business” 1. Replicative polymerases 2. Translesion DNA synthesis (TLS) polymerases ‘backup’ polymerases not as accurate as the normal replicative polymerase lack exonucleotic proofreading activity 3. DNA repair and recombination 4. Reverse transcriptase Restrained to telomerase in eukaryotes, using a RNA template for DNA synthesis
- 11. 15 different mammalian DNA polymerases Example of specialised polymerase: Terminal deoxynucleotidyl transferase (TDT) is expressed only in lymphoid tissue, and adds random nucleotides to double-strand breaks formed during somatic recombination to promote immunological diversity.
- 12. (post-replicative) DNA mismatch repair corrects errors made by DNA polymerase during DNA replication Defects in DNA mismatch repair have been found in several types of cancer, notably colon cancer, and microsatellite sequences that are either shorter or longer than normal are a hallmark of defective MMR. (before sealing by DNA ligase) MSH2 frequently mutated in hereditary nonpolyposis colon cancer (HNPCC) In order to do this the Up to 1000 bp can be removed mismatch repair machinery distinguishes the newly synthesised strand from the template (parental)
- 13. Double stranded breaks environment endogenous • Ionizing radiation DSBs are created biologically by the protein SPO-11 as the • Replication errors highly regulated initiation of meiotic recombination. • Oxidating agents Only used shortly after DNA replication, during interphase
- 14. Same genes, different alleles Same genes, same alleles
- 15. Homologous recombination Homologous recombination requires DSB of DNA (damage or stalled or broken replication fork), invasion of a homologous dsDNA molecule by a ssDNA end, pairing of homologous sequences, branch migration to form a Holliday junction, and isomerisation of the flanking sequences.
- 18. Rad51, a sequence-independent DNA binding recombinase at the branch point Holliday junction BRCA2 sequesters RAD51 via its BCR repeats and its C-terminal motif, mobilises it to the site of damage and then facilitates the formation of helical RAD51– single stranded DNA nucleoprotein filaments that search for a homologous DNA template. Owen Richard Davies & Luca Pellegrini, Nature Structural & Molecular Biology 14, 475 - 483 (2007)
- 19. ATM protein kinase • DSBs create changes in chromatin structure which activate ATM by autophosphorylation, which then induces many cellular responses by phosphorylating a vast number of target proteins. • Associate with the BRCA1- associated genome surveillance complex (BASC) • Can phosphorylate p53 also involved in DNA repair and cell cycle arrest.
- 20. The Fanconi anaemia/BRCA pathway • The FANC protein family is involved in the recognition and repair of damaged DNA. The FA complex is activated when DNA stops replicating because of damage. The core complex can associate with BRCA1 and BRCA2. This complex mediates the monoubiquitylation of FANCD2. Activated FANCD2, in turn, is translocated to chromatin and DNA-repair foci. These foci contain the BRCA1 protein and is known to bind directly to RAD51 and to DNA, and to participate in homology-directed DNA repair.
- 22. BRCA1, in combination with BARD1, has E3 ubiquitin ligase activity; BRCA2 has no enzymatic activity. Both BRCA1 and BRCA2 are tumour suppressor proteins. They form multiple protein complexes with overlapping functions.
Editor's Notes
- The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophage (virusesthat infect bacteria); however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms.[47]
- A key step is an enzyme-mediated “flipping-out” of the altered nucleotide from the helix, which allows the enzyme to probe all faces of the base for damage (Figure 5-51). It is thought that DNA glycosylases travel along DNA using base-flipping to evaluate the status of each base pair. Once a damagedbase is recognized, the DNA glycosylase reaction creates a deoxyribose sugar that lacks its base. This “missing tooth” is recognized by an enzyme called AP endonuclease, which cuts the phosphodiester backbone, and the damage is then removed and repaired (see Figure 5-50A). Depurination, which is by far the most frequent type of damage suffered by DNA, also leaves a deoxyribose sugar with a missing base. Depurinations are directly repaired beginning with AP endonuclease, following the bottom half of the pathway in Figure 5-50A. he second major repair pathway is called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. Such “bulky lesions” include those created by the covalent reaction of DNA bases with large hydrocarbons (such as the carcinogen benzopyrene), as well as the various pyrimidine dimers (T-T, T-C, and C-C) caused by sunlight. In this pathway, a large multienzyme complex scans the DNA for a distortion in the double helix, rather than for a specific base change. Once a bulky lesion has been found, the phosphodiester backbone of the abnormal strand is cleaved on both sides of the distortion, and an oligonucleotide containing the lesion is peeled away from the DNA double helix by a DNA helicase enzyme. The large gap produced in the DNA helix is then repaired by DNA polymerase and DNA ligase (Figure 5-50B).
- HR: single-strand invasion, branch migration, limited DNA synthesisIn mycobacteria, NHEJ is much more error prone than in yeast, with bases often added to and deleted from the ends of double-strand breaks during repair.[9] Many of the bacteria that possess NHEJ proteins spend a significant portion of their life cycle in a stationary haploid phase, in which a template for recombination is not available.[8] NHEJ may have evolved to help these organisms survive DSBs induced during dessication