DNA Mutability & Repair PDF

The Mutability & Repair of DNA BT 451 Chapter 10  Introduction High level of accuracy in DNA synthesis (1 mistake in 1010) Base-pair geometry & complementarity prevent mistakes to occur in 105 times. How mistakes are prevented in order to reach the high l...

The Mutability & Repair of DNA BT 451 Chapter 10  Introduction High level of accuracy in DNA synthesis (1 mistake in 1010) Base-pair geometry & complementarity prevent mistakes to occur in 105 times. How mistakes are prevented in order to reach the high level of accuracy?  Topics Types of DNA errors – SNP – Microsatellite – Transposition – Double strand break Sources of errors 1. Inaccuracy of DNA replication 2. Chemical/radiation damage of DNA 3. Insertion of DNA element (transposons) 4. Spontaneous damage Repair of DNA damage – How DNA is repaired quickly???  Mutation A mismatch will be a mutation in the second round of replication Mutation could be in coding or non-coding DNA  Types of DNA errors (Nature of Mutations) 1. Point Mutation (SNP) Transition Transversion Insertion Deletion  Types of DNA errors (Nature of Mutations) 2. DNA microsatellites Mutation-prone- sequences Insertion/deletion of Di, tri, tetranucleotide sequence (CACACA, CGGCGGCGGCGG) Hot spot areas for mutation Used as physical genetic markers for mapping genetic diseases  Types of DNA errors (Nature of Mutations) 3. Transposition  Types of DNA errors (Nature of Mutations) 4- Double strand break Mainly caused by environmental factors Both strands of the DNA are broken If not repaired can lead to serious consequences Some replication errors escape proofreading 1. Inability of DNA polymerase to add new bp next to wrong one 2. Activation of exonuclease  Mismatch Repair System removes errors that escape proofreading MRS Challenge – Scan the genome to detect errors – Fix these errors accurately before the second round of cell division MRS in E.coli : mismatch repair protein is MutS , a dimer that recognizes the mismatch from the distortion of the DNA  1. MutS Scan DNA 2. Recognize mismatch Recruit MutL Activate MutH 1. Helicase (UvrD) 2. exonucleases Enzyme create an 1. Polymerase III incision 2. Ligase  How can MRS recognize which nucleotide is the mismatched one? E.coli tags the parental strand by transient hemimethylation Dam methylase: methylate A residues on both strands of the sequence 5’GATC3’ This sequence is widely distributed along the entire genome and all of these sites are methylated by the Dam methylase MutH binds to nonmethylated sites but become activated when binds to MutS and MutL Directionality in mismatch repair: Exonuclease removal of mismatched DNA  Eukaryotic MRS MutS & MutL homologs are used (MSH, MLH and PMS) Multiple MutS like proteins with different specificities They lack the MutH and hemimethylation trick used by E.coli Lagging strand synthesis: prior to the ligation step, the Okazaki fragment is separated from previously synthesized DNA by a nick, which can be thought of as being equivalent to the nick generated by the MutH on the newly synthesized strand Human homolog of MutS (MSH) interact with the sliding clamp component of the replisome and would be recruited to the site of discontinuous DNA synthesis on the lagging strand. This interaction could recruit mismatch repair system  Mismatch repair system in Eukaryotics E. coli MutS MutL Eukaryotics MSH MLH or PMS (MutS homolog) Hereditary nonpolyposis colorectal cancer (mutations in human homologes of Muts and MutL)  Sources of errors 1. Spontaneous damage 2. Environmental factors 3. Inaccuracy of DNA replication 4. Insertion of DNA element (transposons) Ames test for mutagens  1. Spontaneous Damage A- damage caused by water (hydrolysis) Deamination of C → U Deamination of A → hypoxanthine can H-bond to C Deamination of G → xanthine can H-bond to C (two H-bonds) Methylated C are hotspots for spontaneous mutations in vertebrate DNA 2. Depurination Hydrolysis of N-glycosyl linkage → abasic site (apurinic)  2. Damage caused by environmental factors a. Alkylation b. Oxidation c. Radiation – UV light – Gamma ray – X ray  2. Damage caused by environmental factors A- Alkylation Methyl/Ethyl group added to base or DNA backbone Nitrosamines and N-methyl-N1-nitrosoguanidine O atom of guanine C6 → O6-methylguanine T  Damaged by alkylation and oxidation Alkylation at the oxygen of carbon atom 6 of G : O6- metylguanine, often mispairs with T. Oxidation of G generates oxoG, it can mispair with A and C. a G:C to T:A transversion is one of the most common mutation in human cancers.  2. Damage caused by environmental factors B- Oxidation Ionizing radiation or chemicals producing free radicals Caused by oxidizing agents (O2–, H2O2, OH ) Oxidation of guanine → oxoG A C This transversion is very common in cancer  2. Damage caused by environmental factors C- UV radiation Two adjacent pyrimidines (T/C) join together Photochemical fusion cause DNA polymerase to stop  2. Damage caused by environmental factors D- gamma & X-ray radiation Gamma radiation used for gamma camera & gamma knife X ray used to radiograph bone to detect fractures /deformity Cause double strand break by ionizing DNA backbone Also it forms free radicals Cell can’t divide when chromosomes are broken → apoptosis Used for chemo/radio therapy Clastogenic: ionizing radiation and agents like bleomycin that cause DNA to break  Damage caused by base analogs T analog Structurally similar to the base that can be taken by the cell and incorporated with DNA Mutations caused by intercalating agents Intercalating agents flat molecules Causing addition or deletion of bases during replication  Spontaneous Damage damage caused by intercalating agents Flat molecules of polycyclic rings bind to DNA Proflavin, acridine and ethidium Cause insertion/deletion of one or a few bp How? Skipping or addition of new bp. In case of insertions , by slipping between the bases in the template strand, these mutagens cause DNA polymerase to insert an extra base opposite the intercalated molecule In case of deletion, the distortion to the template caused by the presence of an intercalated molecule might cause the polymerase to skip a nucleotide Serious consequence on translation of mRNA  DNA Repair G1 & G2 are check points before replication and division Cell aim to repair any damage to pass to M phase In all previous methods template DNA was available Repair of DNA Damage: DNA repair system Excision repair systems: the damaged nucleotide is not repaired but removed from the DNA, the other undamaged strand serves as a template for reincorporation of the correct nt by DNA polymerase Recombination repair: both strands are damaged. Sequence information is retrieved from a second undamaged copy of the chromosome.  1- Direct reversal of DNA damage Repair of damage caused by UV radiation photoreactivation  1- Direct reversal of DNA damage O6-methylguanine is repaired directly by methyl transferase.  2- Repair of hydrolysis damage base excision repair system Glycosylase enzyme – Recognize damaged base – hydrolyze glycosidic bond – Repair is completed by Pol & ligase – Different specificities (11 different ones in human)  Base excision pathway: The uracil glycosylase reaction  Base excision repair DNA glycosylases are lesion-specific and cells have multiple DNA glycosylases 1. Uracil glycosylase 2. Another specific glycosylase is responsible for removing oxoG AP: apurinic or apyrimidinic  How can Glycosylase complex recognize abnormal base? Evidence indicates that these enzymes Diffuse laterally along the minor groove of DNA until a specific lesion is detected Damaged base is flipped out  Fail-safe glycosylase oxoG: A reapir Works in case glycosylase did not recognize the error before replication Fail-safe system that removes T opposite a G (arise from deamination of 5-mehylcytosine  3- Nucleotide excision repair (NER) cleaves damaged DNA on either side of the lesion Recognize DNA distortion (T dimers or bulky adduct on a base) Short single-stranded segment is removed 4 proteins involved in E.coli: UvrA UvrB UvrC UvrD  The principle of NER in higher cells is much the same as that in E.coli but more complicated XPC (detect distortion of DNA like UvrA) Formation of bubble involves the helicase activities of proteins XPA and XPD (equivalent of UvrB in E.coli) and the SSB protein RPA. The bubble creates cleavage sites on the 5’ side of the lesion for a nuclease ERCC1-XPF on the 3’ side for the nuclease XPG (like UvrC) Resulting ssDNA segment is 24-32 nucleotide long. Nucleotide excision repair enzyme UVR proteins recognize broad variety of changes including dimmers caused by UV Mutants to UVR proteins are sensitive to UV light Xeroderma pigmentosum (7 XP genes)  Nucleotide Excision Repair The principles of nucleotide excision repair in higher cells is much the same as in E. coli but us moer complicated, involving 25 or more polypeptides. The UVR proteins are needed to mend damage from UV light; Mutants of uvr genes are sensitive to UV light, and lack the capacity to remove T-T or T-C adducts. In human, xeroderma pigmentosum patients have mutations in seven genes (XP genes). These XP proteins are corresponding to proteins involved in nucleotide excision repair.  4- Transcription- coupled repair RNA polymerase serve as damage sensing protein Stop RNA polymerase and call nucleotide excision repair proteins Has the advantage of focusing on genes being transcribed Transcription - coupled repair in Eukaryotes TFIIH – Includes XPA & XPD subunits – Unwind DNA template during transcription – Melt the DNA around the lesion during nucleotide excision repair/ transcription coupled repair 5- Double strand break (DSB) repair. Recombinational repair Both strands are broken (no template) Sister chromosome is a template Helps repair errors in DNA replication  What happens if the break took place before replication (no sister chromosome)? Fail safe method nonhomologous end joining (NHEJ), a backup system in yeast Two broken DNA ends joined together. Because the sequence information is lost from the broken ends, the original sequence is not restored (its mutagenic but less hazardous to the cells than unrepaired broken ends) 7 components found in mammalian cells: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, Cernunnos-XLF, and DNA ligae IV Ubiquitous in eukaryotic cells, les frequent in bacteria Not efficient method except in spores of B. subtilis (has a two-protein NHEJ system that repairs DNA breaks when the spore germinates)  6- Translesional DNA synthesis Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the site of the damage. In E. coli, DNA Pol IV (DinB) or DNA Pol V (a complex of the proteins UmuC and UmuD`) performs translesion synthesis. DinB and UmuC are members of a distinct family of DNA polymerases found in many organisms known as the Y family of DNA polymerases There are five translesion polymerases known in humans, four of which belong to the Y family. Y-Family DNA polymerases: specialize in translesion synthesis, bypassing damaged bases that would otherwise block the normal progression of replication forks. Y-Family polymerases are also characterized by a low catalytic efficiency, a low processivity, and a low fidelity on normal DNA. Translesion DNA synthesis: although are template dependent, the synthesis in a manner that is independent of base pairing. obstacles to progression of the DNA polymerase (or AP site) Complex of proteins UmuC and D’ (Y-family of DNA polymerase) Box 10-6 Y-Family DNA polymerases  In E. coli, the translesion polymerases are not present under normal circumstances. Rather, their synthesis is induced only in response to DNA damage. Thus, the genes encoding the translesion polymerases are expressed as part of a pathway known as the SOS response. Damage leads to the proteolytic destruction of a transcriptional repressor (the LexA repressor) that controls expression of genes involved in the SOS response, including those for DinB, UmuC, and UmuD, the inactive precursor for UmuD`. Interestingly, the same pathway is also responsible for the proteolytic conversion of UmuD to UmuD`. Cleavage of both LexA and UmuD is stimulated by a protein called RecA, which is activated by single-stranded DNA resulting from DNA damage. RecA is a dual-function protein that is also involved in DNA recombination.  How a translesion polymerase gains access to the stalled replication machinery at the site of DNA damage? In mammalian cells, entry into the translesion repair pathway is triggered by chemical modification of the sliding clamp (attachment of ubiquitin). Once ubiquitinated, the sliding clamp recruits translesion polymerase through ubiquitin. The translesion polymerase somehow displaces the replicative polymerase from the 3’ end of the growing strand and extends it across the site of damage. Alternative models for translesion synthesis: a- The polymerase-switching model b- The gap-filling model END