Mahantesha - Master Seminar On MAHANTHES HA M 2 Mutability...

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Unformatted text preview: Master Seminar On MAHANTHES HA M. 2 Mutability and DNA Repair The perpetuation of the genetic material from generation to generation depends on maintaining rates of mutation at low levels High rates of mutation would destroy the species in the germ line and the individual in the soma At the same time ,if the genetic material were perpetuated with perfect fidelity the genetic variation needed to drive evolution would be lacking ,and new species would not arisen Mutation is the ultimate source for creation of new variability Life and biodiversity depend in a happy balance between mutation and its repair OUTLINE Foreword Replication errors and their repair DNA damage Repair of DNA damage Case study Something unknown Summary Foreword 1.Mutation Sudden heritable changes in a characteristics of an organism Type Point mutation is the mutation that alter a single nucleotide Transitions (pyrimidine to pyrimidine, purine to purine) Transversions (pyrimidine to purine, purine to pyrimidine) Insertions Deletions Gross rearrangement of chromosome “Hotspots” Sites on the chromosome where the mutations arise at high frequency. (DNA microsatellite, Mutation-prone sequence in human genome are repeats of simple di, tri or tetra-nucleotide sequences) These sequences 1) Important in human genetics and disease 2) Hard to be copied accurately and highly polymorphic in the population 2.Three important sources for mutation (unavoidable) 1. Inaccuracy 2. Chemical 3. The in DNA replication damage to the genetic material insertion generated by DNA elements known as transposition 3.Two consequences of mutation unrepaired (1) Permanent changes to DNA which can alter the coding sequence of a gene or its regulatory sequence (2) Some chemical alterations to DNA prevent its use as a template for replication and transcription So Repair of DNA is important to the organisms Replication errors and their repair The proofreading mechanism ( the 3’→5’ exonuclease component) improves the fidelity of DNA replication, but it is not foolproof So, there are a total of 12 possible mismatches during the DNA replication (T:T,T:G,T:C, and so forth) The mis-incorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation Mismatch repair removes errors that escape proofreading Process of repair in detail. Firstly, E. coli’s repair system 1.How to scan the mismatches and remove errors? 2.How to distinguish the mismatched strand and the parental strand? 1.How to scan the mismatches and remove errors? MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone MutS is a dimer of the mismatch repair protein MutS embraces the mismatchcontaining DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself MutS-mismatchcontaining DNA complex recruits MutL MutL activates MutH, an enzyme causing an incision or nick on one strand near the site of the mismatch MutL is a second protein component of the repair system MutH is an enzyme causing an incision or nick on one strand Helicase (UvrD) unwinds the DNA starting from the incision and moving in the direction of the site of the mismatch Exonucleases progressively digests the displaced single strand extending to and beyond the site of the mismatched nucleotide This action produces a single-stranded gap, which is then filled in by DNA polymerase Ⅲ and sealed by DNA ligase 2.How to distinguish the mismatched strand and the parental strand? The answer is that E. coli tags the parental strand by transient hemi - methylation as we now describe The GATC sequence is widely distributed along the entire genome ,and all of these sites are methylated by the Dam methylase So ,before the newly strand is methylated by the Dam methylase after the DNA replication ,the resulting daughter DNA duplexes will be hemimethylated Thus the newly strand is marked (it lacks a methyl group) and hence can be recognized as the strand for repair Different exonucleases are used to remove ssDNA between the nick created by MutH, depending on whether MutH cuts the DNA in the 5’ or 3’ side of the misincorporated nucleotide Secondly, the Eukaryotic cells repair system The Eukaryotic cells repair mismatches and do so using homologous to MutS (MSH) and MutL (MLH) But they lack MutH and E.coli’s clever trick of using hemimethylation to tag the parental strand How does the mismatch repair system of the Eukaryotic cells know which of the two strands to correct ? It takes place discontinuously with the formation of Okazaki fragments that are joined to previously synthesized DNA by DNA ligase Prior to the ligation step ,the Okazaki fragment is separated from previously synthesized DNA by a nick created, which can be though of as being equivalent to the nick created in E.coli by MutH on the newly synthesized strand DNA damage DNA undergoes damage spontaneously from hydrolysis and deamination DNA damaged by alkylation , oxidation and radiation Mutations are also caused by base analogs and intercalating agents 1.Hydrolytic damage Deamination of the base cytosine (just show in the picture on the left) In contrast to the replication errors ,all of these hydrolytic reactions result in alterations to the DNA that are unnatural 2.DNA damaged by alkylation , oxidation and radiation Alkylation In alkylation ,methyl or ethyl groups are transferred to reactive sites on the base and to phosphates in the DNA backbone Oxidation DNA is also subject to attack from reactive oxygen species . (eg. O2-, H2O2, and OH•) ■ Radiation Ultraviolet light ,which produce the photochemical fusion of two pyrimidines that occupy adjacent positions on the same polynucleotide chain. (eg. thymine dimer) These linked bases are incapable of base-pairing and cause the DNA polymerase to stop during replication Gamma radiation and X-ray, which cause double-strand breaks in the DNA ,which are difficult to repair 3.caused by base analogs and intercalating agents Base analogs Similar enough to the normal bases to be processed by cells and incorporated into DNA during replication But their base pair different,leading to mistake during replication Intercalating agents They are flat molecules containing several polycyclic rings These rings bind to the equally flat purine or pyrimidine bases of DNA, just as the bases bind or stack with each other in the double helix Repair of DNA damage DNA repair system Direct reversal of DNA damage Excision repair system Recombination (DSB) repairs Translesion DNA synthesis 1.Direct reversal of DNA 1. damage A repair enzyme simply reverses (undoes) the damage Two examples in detail to understand the direct reversal of DNA damage (1).Photoreactivation The enzyme DNA photolyase captures energy from light and it to break the covalent bonds linking adjacent pyrimidines So, the damaged bases are mended directly (2).The removal of the methyl group The methyltransferase removes the methyl group from the methylated 6methylguanine The methyl group is transferred to the protein itself, inactivating the protein(very costly) 2. Excision repair system Two kinds of excision repair exist, Base excision repair Nucleotide excision repair (1).Base Excision Repair Removal of only the damaged nucleotide An enzyme called as glycosylase (lesiong specific) recognizes and removes the damaged base by hydrolyzing the glycosidic bond The resulting basic sugar is removed from the DNA backbone After the damaged nucleotide has been entirely removed from the backbone, a repair DNA polymerase and DNA ligase restore an intact strand using the undamaged strand as a template Removes the damaged base and repair What if a damaged base is not removed by base excision before DNA replication ? There are some fail-safe systems to deal with this problem There are two examples in detail oxoG mispair with A A dedicated glycosylase which recognizes the oxoG:A base pairs It recognizes an A opposite an oxoG as a mutation and removed the undamaged but incorrect base T mispair with G A glycosylase removes T from T:G mispairs The glycosylase system assumes that the T in the T:G mismatch arose from deamination of 5-methyl-cytosine and selectively removes the T so that it can be replaced with a C (2). Nucleotide Excision Repair Removal of a short stretch of single-strand DNA that contains the lesion These NER enzymes do not recognize any particular lesion, they work by recognizing distortions to the shape of the double helix Such distortions trigger a chain of events that lead to the removal of short singlestranded segment which is filled in DNA polymerase using the undamaged strand as a template The nucleotide excision repair of n E.coli (a)UvrA and UvrB scan DNA to identify a distortion (b) UvrA leaves the complex ,and UvrB melts DNA locally around the distortion (c)UvrC forms a complex with UvrB and creates nicks to 5’ side of the lesion and to the 3’ side of the lesion . (d)DNA helicase UvrD releases the single stranded fragment from the duplex ,and DNA pol 1and ligase repair and seal the gap Transcription-coupled repair Nucleotide excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template 3. Recombination (DSB) repairs How do cells repair double-strand breaks in DNA in which both strands of the duplex are broken ? 1.When the sister of the broken chromosome is present in the cell , the DSB-repair (double strand break system) pathway can operate the DSB-repair retrieves sequence information from the sister chromosome 2. When a chromosome break early in the cell cycle, before a sister has been generated by DNA replication , a fail- safe system comes into play known as NHEJ (non homologous end joining) NHEJ does not involve homologous recombination Instead, the two ends of broken DNA are directly joined to each other by misalignment between single strand protruding from the broken ends 4. Translesion DNA synthesis If cells cannot repair some lesions, there is a fail-safe mechanism that allows the replication machinery to bypass these sites of damage. This mechanism is known as translesion synthesis But because of its high error rate translesion synthesis can be considered a system of last resort Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the site of the damage Suzanne et al (2008) In brief....... CASE STUDY Ozone depletion in the stratosphere has resulted in increased UV-B radiation at the earth's surface, which induces various DNA lesions. The major lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidinone dimers photoproducts The minor lesions: oxidized or hydrated bases, singlestrand breaks Through evolution, plants have acquired two main protective Strategies: 1. The shielding by flavonoids and phenolic compounds 2. The DNA repair such as photoreactivation and dark repair Photoreactivation: Mediated by photolyase, the major DNA repair pathway for CPDs and photoproducts in higher plants Photolyases bind specifically to these DNA lesions and remove them directly by absorbing light in the 300±600 nm range The dark repair includes: nucleotide excision repair (NER), base excision repair (BER), mismatch repair NER sequentially involves: 1. Recognition of DNA damage 2. Incision on damaged strand 3. Excision of damage containing oligonucleotides 4. DNA synthesis and ligation There are two sub pathways of NER: a) Designated global genomic repair (GGR): repairs the DNA damage over the entire genome b) Transcription-coupled repair (TCR): selective for the transcribed DNA strand in expressed genes Oxidized or hydrated bases and single-strand breaks are repaired by BER. 1. DNA glycosylases initiate this process by releasing the damaged base 2. With cleavage of the sugar phosphate chain 3. Excision of the basic residue containing oligonucleotides 4. DNA synthesis and ligation There are two sub pathways for BER. a) The short patch BER is DNA polymerase beta-dependent b) while the long-patch BER is DNA polymerase delta/epsilon-dependent Excision repair (NER and BER) is very important for maintaining genome stability and essential for survival for organisms Seisuke et al (2004) Sesuke et al.(2004), Investigated several plant genes related to DNA repair and replication The expression levels of excision repair genes were found to be high in meristematic tissues • Expression patterns of plant genes for dark repair analyzed by Northern hybridization In situ hybridization Oligo DNA microarray analysis In vivo DNA repair assay Results: Photoreactivation is the main pathway for removal of DNA damage in mature leaves (non-proliferating cells) and dark repair is coordinated with cell proliferation Seisuke et al (2004) Something unknown Although the repairs of damaged DNA have formed systems, there are still a lot of problems which have not been solved and need to be studied more deeply For example: The mechanism by DNA glycosylase g scan for damaged bases remains mysterious In translesion synthesis 1.How does the translesion polymerase recognize a stalled replication fork ? 2.How does the translesion enzyme replace the normal replication polymerase in the DNA replication complex? 3.Once DNA synthesis is extended across the lesion ,how does the normal replication polymerase switch back to and replace the translesion enzyme at the replication fork? Summary The main content of this presentation : Errors that are generated during replication, lesions that arise from spontaneous damage to DNA The damage that is brought by chemical agents and radiation In my opinion , in each case we should consider, What cause the alteration to the genetic material ? How the alteration to the genetic material is detected ? How it is properly repaired ? Then if we understand these problems clearly, we must make it! THANK YOU 60 ...
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