Molecular Genetics IV.pdf

Molecular Genetics IV DNA Repair Molecular Biology Source of mutation Type of mutation Type of mutation Reactive Oxygen Species Chemical Changes Base Excision repair (BER) U.V. Light Thymine-Thymine dimers DNA replication Replication errors Nucleotide Excision Repair (NER) Proof-reading Mismatch Rep...

Molecular Genetics IV DNA Repair Molecular Biology Source of mutation Type of mutation Type of mutation Reactive Oxygen Species Chemical Changes Base Excision repair (BER) U.V. Light Thymine-Thymine dimers DNA replication Replication errors Nucleotide Excision Repair (NER) Proof-reading Mismatch Repair (MMS) NHEJ Radiation Double strand breaks Homologous Recombination (HR) Defects in DNA repair mechanisms and cancer are closely related. When repair mechanisms are compromised, mutations accumulate in the cell’s DNA. If these mutations affect genes that are normally involved in the careful regulation of cell division, cells can begin to divide uncontrollably, leading to tumor formation, and cancer. Chemically modified bases DNA is continually subjected to a barrage of damaging chemical reactions; estimates of the number of DNA damage events in a single human cell range from 104 to 106 per day! For example, the bond connecting a purine base to deoxyribose is prone to hydrolysis at a low rate under physiological conditions, leaving a sugar without an attached base. Reactive Oxygen Species (ROS) like hydroxyl radicals and peroxide groups are natural byproducts of cell metabolism and can chemically change nucleotide bases. Example: change from C to T through deamination. Base Excision Repair (BER) This mechanism removes chemically modified bases and prevents point mutations after replication BER mechanism Steps: 1.Identify the modified base and mark it with a sugar: glycosylation by DNA glycosylase. 2. Cleave the base off by APEI endonuclease 3. Bring the right base and ligate it to the rest of the strand (DNA polymerase + DNA ligase) Thymine-Thymine dimers U.V. light induces the formation of Thymine-thymine dimers These dimers interfere with both replication and transcription of DNA. Nucleotide Excision Repair (NER) This is the main mechanism that repairs T=T dimers. Over 30 proteins are involved in this pathway. Steps: 1. Recognition of the dimer 2. Opening of the DNA duplex (XPC) 3. Excision of a region that includes T=T dimer (XP-F and XP-G) 4. Polymerize a new strand and ligate it to the existing one Xeroderma pigmentosum Genes involved in NER were identified through a study of the defects in DNA repair in cultured cells from individuals with Xeroderma pigmentosum. That explains why they are named as XPXeroderma pigmentosum, a hereditary disease associated with a predisposition to cancer. Individuals with this disease frequently develop the skin cancers called melanomas and squamous cell carcinomas if their skin is exposed to the UV rays in sunlight Cells of affected patients lack a functional nucleotide excision-repair system. Replication errors Replication errors are quite common: In E.coli 1 base every 10.000 is wrongly match during replication. Yet mutation rate are only 1 every 100 million bases, Why? Proof-reading activity of the DNA polymerase, which acts as the first line of defense DNA polymerase contains a 5-to-3 DNA polymerase activity and a 3-to-5 exonuclease activity or proofreading Replication errors The second line of defense for replication errors is the Mismatch Repair Pathway (MMR) which fixes mistakes that escaped the proofreading activity of the DNA polymerase STEPS: 1. Recognize the mismatch and determine which strand is wrong (done by MSH2 and MSH6) 2. Remove a chunk of the wrong strand by MLH1 endonuclease 3. Gap repair by DNA polymerase and DNA ligase Double strand breaks (DSB) The most serious form of DNA damage. Of left unrepaired can lead to translocations and genome instability Can be produced by ionizing radiation or stalled replication forks that collapse Double strand breaks (DSB) Can be repaired by: Non-homologous end Joining (NHEJ) -Prone to error -Most common form in mammalian Homologous recombination (HR) -More accurate -Only possible when homologous sequences are available -Mammalian cells only used in Late S-phase and G2 NHEJ Straight joining of both ends of a DSB with common loss of nucleotides STEPS: 1. Recognition of DSB by Ku 2. Each Ku at each end bind to each other and ‘bridges’ the two ends 3.Recruitment of DNA-PKcs by Ku. 4.DNA-Pkcs replaces Ku as the the protein that ‘bridges’ the two ends 5. Recrutiment of XLF-XRCC4 and Ligase IV complex to ligate the two ends HR Several human cancers are potentiated by inherited mutations in genes essential for homologous recombination repair For example, some women with an inherited susceptibility to breast cancer have a mutation in one allele of either the BRCA-1 or the BRCA-2 genes that encode proteins participating in this repair process. This mechanism involves an exchange of strands between separate DNA molecules and hence are referred to as DNA recombination. HR: repair of double strand break (DSB) When a DSB forms, HR can repair it using the homologous sequence. The first step is digestion of the 5’ end to leave highly recombinogenic 3’ ends HR: repair of double strand break (DSB) Second Step: Strand invasion mediated by Rad51 (RecA in bacteria) DSBR: Double strand break repair SDSA: Synthesis dependent Strand annealing HR: repair of double strand break (DSB) Third Step: Progression of the Fork HR: repair of double strand break (DSB) Next Step: Resolution of the Holiday Junction -Cleavage and ligation Holiday Junction HR: repair of double strand break (DSB) There are multiple ways to resolve the Holiday junction and each way will produce different products Gene conversion or crossover Depending of the way the HJ is resolve one can have gene conversion or a crossover that can lead to a translocation A B a b a B A b a b a B