BIMM 100 Lecture 5

BIMM 100 Lecture 5 - BIMM100 Lecture 5: Using...

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Unformatted text preview: BIMM100 Lecture 5: Using molecular biology ­ recombinant DNA Read: Lodish pages 165 ­176 (for a review), and 176 ­186 Also read the two classic experiments (reverse transcriptase & restricMon enzymes) posted on Ted Lecture summary •  DNA replicaMon –  –  –  –  The process The players CoordinaMon and bi ­direcMonal replicaMon Finishing •  DNA mistakes, damage, and repair –  Polymerase ­ errors and proofreading –  Damage and repair •  •  •  •  DeaminaMon Base excision Mismatch repair UV damage and nucleoMde excision repair –  Global vs. transcripMon coupled •  Non homologous end joining ­ the last chance •  Viruses •  Recombinant DNA Finish up our discussion of DNA repair and viruses GeneMcs: terms and concepts ­review it! Cloning! Base excision (to repair T ­G mismatches and damaged bases) ­happens before replicaMon! Problem: which strand is normal? Which strand is mutant? Since the deaminaMon of C to T is so common, this repair system has evolved to remove T, replacing it with a C. Glycosylase “flips” out the T, and hydrolyzes the bond that connects it to the sugar phosphate backbone, effecMvely “cu`ng” it out, leaving only a deoxyribose. APE1 (apurinic endonuclease 1) is an endonuclease specific for the “baseless” site. It cuts the DNA backbone DNA Polβ is a special repair polymerase AP lyase removes the deoxyribose Mismatch excision repair (for uncorrected polymerase errors that occur during replicaMon) Again ­ the problem ­ which strand is good? Which is mutant? The machinery somehow recognizes the daughter strand, and assumes it is the mutant copy. 1. MSH2 and MSH6 complex and bind mismatch 2. MLH1 and PMS2 bind, and recruit a DNA helicase and endonuclease to cut the daughter strand of DNA (unknown method!) The helicase unwinds the DNA, and exonucleases degrade the daughter strand. 3. The gap is filled by DNA polymerase and the ends are ligated. Mismatch excision repair (afer replicaMon!) MER is also used to repair small inserMons and deleMons in the genome! Autosomal dominant inheritance MutaMons in these genes are Med to hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome (named afer Henry Lynch, who characterized it in 1966). PaMents with inherited mutaMons in their DNA repair machinery have: 80% chance of ge`ng colon cancer in their lifeMme 80% chance of ge`ng endometrial cancer in their lifeMme High chance of ge`ng urinary tract and bowel cancers. UV damage causes a specific type of DNA damage: pyrimidine dimers (can also happen with C!) UV radiaMon damage is common! DistorMon of the DNA duplex Intra ­strand covalent link Cyclobutane pyrimidine ring How is this repaired? NucleoMde excision repair (NER) 1. Complex of XP ­C and 23B proteins recognize lesion 2. They recruit TFIIH, which has helicase subunits (ATP hydrolysis!) that unwinds the DNA. XP ­G and RPA also bind to help unwind and stabilize the “bubble” of about 25 bases. 3. XP ­G (now funcMons as a nuclease) and XP ­F cut the damaged DNA strand at points 24 ­32bp from the lesion. That piece is removed and immediately degraded into nucleoMdes. 4. DNA polymerase fills the gap, and ligase seals the ends. Lack of nucleoMde excision repair has serious clinical consequences! A dose of UVB radiaMon sufficient to induce erythema (sunburn) results in the formaMon of about 105 cyclobutane pyrimidine dimers (CPDs) per epidermal cell! Transgenic mice engineered to lack these repair mechanisms are prone to inflammaMon and edema. UVB exposure locally introduces DNA damage in epidermal keraMnocytes and immune cells, which go on to produce inflammatory cytokines, beginning a general cascade of immune responses in the organism. Under circumstances that DNA repair is decreased or delayed, mutagenicity is enhanced (ex: xeroderma pigmentosum paMents). This disease is characterized by elevated suscepMbility to sunburn and high incidence of skin cancer. In a randomized study, the topical applicaMon (daily for 1 year) of the DNA repair enzyme T4 endonuclease V to XP paMents reduced the rate of appearance of new acMnic keratoses and basal ­cell carcinomas, implying that the reducMon of persistent CPD protects against the development of skin cancer. VariaMon on the same process: transcripMon coupled repair (TCR) RNA polymerase is stalled at lesion CSB is recruited, which triggers opening of DNA helix at that point ­ then, TFIIH, RPA, and XP ­G are recruited to finish the job! EssenMal, because this directs repair to criMcal regions (regions being transcribed) so mutaMons will not be generated! CSB (also called ERCC ­6): named afer Cockayne syndrome B Rare autosomal recessive congenital disorder associated with impaired growth, impaired development of the nervous system, sensiMvity to sunlight, and premature aging. Non ­homologous end joining (NHEJ): for serious DNA damage Joining of non ­homologous ends to repair double ­stranded breaks 1. Ku and DNA dependent protein kinase (PK) bind free ends of the double stranded break. Ku’s helicase unwinds both free ends. 2. Synapse is formed, and ends are processed by nucleases, removing bases from the DNA ends. Other proteins: Rad50, MRE11, and NBS11 complex chews off the damaged ends. 3. Double stranded breaks are ligated back together. Ends are joined, but some basepairs are lost ­ this process is error prone! Lecture summary •  DNA replicaMon –  –  –  –  •  The process The players CoordinaMon and bi ­direcMonal replicaMon Finishing DNA mistakes, damage, and repair –  Polymerase ­ errors and proofreading –  Damage and repair •  •  •  •  DeaminaMon Base excision Mismatch repair UV damage and nucleoMde excision repair –  Global vs. transcripMon coupled •  Non homologous end joining ­ the last chance •  Viruses •  Recombinant DNA –  Host range –  LyMc vs. non ­lyMc –  Retroviruses and reverse transcriptase Viruses (overview) •  Intracellular parasites –  Cannot reproduce by themselves ­ they must commandeer a host cell’s machinery to help them make proteins and replicate their genome •  Specific host range ­limited species infected –  Some only have receptors for certain cell types •  MulMple mechanisms of replicaMon and propagaMon –  RNA and DNA genomes •  Important for human, animal, and plant health Viruses: transmission of biological informaMon transcripMon Other methods of transmi`ng biological informaMon DNA translaMon RNA DNA replicaMon Retroviruses like rotavirus [dsRNA], polio [ssRNA], and HIV [ssRNA]. Reverse transcripMon RNA replicaMon (in certain viruses) PROTEIN How to culture and count virus parMcles: the plaque assay Plaque: an absence of cells (i.e. presence of a virion) The lyMc life cycle (non ­enveloped, bacterial virus) DNA virus ­ the host cell transcribes genes from the bacterial genome, and then translates them into proteins. These “early” proteins replicate the viral DNA and induce expression of “late” viral genes, essenMal for packaging and degradaMon of the host cell. Eventually the cell dies, spewing virus to infect other cells Viruses: many flavors •  MulMple modes of “life cycles” –  LyMc: infected cells are lysed –  Non ­lyMc: cells are not lysed. Usually the viral genome is integrated into the host genome •  Examples: –  Bacterial λ phage –  Human DNA viruses, like human papilloma virus (HPV ­ causes warts and cervical cancer) –  Human retroviruses, like HIV and human t ­lymphotropic virus (HTLV) Retroviruses: the life cycle CRITICAL! ­know this classic experiment! Retroviral infecMon: RNA genome is copied by viral RT enzyme into dsDNA Integrated into the host genome Stably replicated (and segregated) by the host cell machinery Packaged and secreted to infect other cells! Animated cycle of retroviral infecMon See two other animaMons, too! Pharmacology: how to treat retroviral infecMons •  Case study: HIV –  Current therapy includes a “cocktail” of anMretroviral agents •  Nucleoside Analog Reverse Transcriptase Inhibitors (NRTIs): nucleic acid analogs that disrupt the synthesis of viral DNA synthesis by RT. They get integrated and terminate the DNA chain. •  Protease inhibitors (Pis): inhibit protease, and enzyme that HIV uses to cleave nascent proteins for the final assembly of new virions. •  Non ­nucleoside Reverse Transcriptase Inhibitors (NNRTIs): directly block RT acMvity •  Entry inhibitors (new, used for paMents generally infected with viruses resistant to common therapies): prevent viral fusion, uptake, and entry. •  Integrase inhibitors: block integrase, an essenMal enzyme that integrates the viral DNA into the cell’s DNA. Several under clinical trial. Recombinant DNA •  Overview and geneMc concepts •  Cloning –  Tools –  Building a clone •  Libraries –  A source of clones –  Screening •  Defining what you’ve cloned Recombinant DNA technology •  A variety of techniques that allow researchers to study an organism’s DNA •  UMlized for DNA cloning –  Simple definiMon? GeneraMng large amounts of iden%cal DNA molecules. •  Stuff you should know: geneMc terms (know pages 165 ­176) –  –  –  –  –  –  –  –  –  Mutant/mutaMons Homozygous/heterozygous Haploid/diploid Recessive/dominant Lethal/condiMonal ComplementaMon Double mutants Suppressor mutaMons GeneMc mapping Recombinant DNA •  Cloning: –  RestricMon nucleases –  DNA ligases –  Vectors (plasmids) •  Libraries –  Building and screening •  Finding a clone/gene –  By sequence –  By funcMon •  Defining a clone –  Describing & sequencing Plasmid cloning: the animated overview What is a clone? •  Clone: fragment of your DNA of interest + a vector •  DNA of interest: preparing for cloning –  RestricMon endonucleases & ligases –  Genomic DNA? Or cDNAs? •  Vector properMes –  Propagated ­ replicated, stably inherited in host cells –  GeneMcally stable ­ has selectable “traits” or “markers” –  Easily manipulated ­ small, easy to amplify –  Some have advantages or “tricks” that can be uMlized to express foreign DNA How can you make a clone? First you need restricMon enzymes… RestricMon enzymes: endonucleases that are produced by bacteria that recognize a certain site of DNA, and cleave it. They are specific! Why? A primiMve host defense in bacteria. The bacterial DNA is protected (by methylaMon of the DNA by a modificaMon enzyme). Foreign DNA is not (from a virus, for example), and is cut out. Leaves compaMble (sMcky) ends Inverted repeat (palindromic sequence) Different types of restricMon enzymes Hundreds of different types! They recognize specific sequences (different recogniMon sites). They cut in different ways (s%cky vs. blunt) RestricMon enzymes: protecMve enzymes in bacteria Problem: EcoRI cuts every 6bp. So, it will cleave DNA at an average of 46 bp (1/4096 bases will be cleaved). This would quickly destroy the bacteria’s DNA! In vivo, the bacteria protects itself ­ by methylaMon of key residues. Now the endonuclease can’t bind, and won’t chop up the DNA! These paired proteins protect the host cells Know classic experiment 5.2 Plasmid vectors: necessary for replicaMng DNA in a bacterial cell (how we “trick” bacteria into making more DNA for research) For replicaMon in E. coli (50 ­100bp) Circular, double ­stranded DNA molecule that is replicated in the host cell (usually E. coli) AnMbioMc resistance ­ ampr encodes β ­lactamase Also known as the mulMple cloning site (MCS) How can we assemble specific DNA fragments into vectors? EcoRI TaqI HindIII These remain in soluMon! Vector and DNA fragments must have compaMble ends. They undergo complementary base pairing. Covalent bonds are formed by DNA ligase (this is dependent on the presence of enzyme and is energy dependent). This reacMon is dependent on the presence of a 5’ phosphate! Blunt end ligaMons are possible, but much less efficient! A generic cloning strategy Vector: prepared by restricMon endonuclease digesMon (“opened up”) Insert: DNA of interest prepared by digesMon or PCR. Usually up to 10kb. LigaMon to generate recombinant plasmid TransformaMon: introducMon of foreign DNA into the “competent” bacterial host cells SelecMon for cells that are replicaMng the recombinant plasmid What are some controls we could do here? Libraries and their role in recombinant DNA technology •  A library is a collecMon of clones •  Genomic DNA vs. cDNA (complimentary DNA that represents copies of mature mRNA in DNA form). –  cDNA lacks noncoding regions (introns) of genes. –  Use polyA tails to obtain and create cDNA library •  Insert size consideraMons/ vector limits –  Plasmids (10 ­20kb max) –  Bacterial arMficial chromosomes (BACs; up to 2Mb [2 million nts]) •  RepresentaMon –  How many clones represent a genome? –  How do we find the clone/sequence of interest? Making a cDNA library in a plasmid vector Isolate mRNA from cells or Mssues Separate mRNA (which has polyA tails) by binding them to oligo ­dT sequences (could be immobilized on beads in a column. Add oligo ­dT primer, and then reverse transcriptase. The RT will transcribe mRNA into cDNA Building a cDNA library in a plasmid vector conMnued Remove RNA (with alkali condiMons or enzymes) Add poly(dG) tail (to hybrize to later) Single stranded cDNA! Hybridize with an oligo ­dC primer (to be able to generate a double stranded version of cDNA) Building a cDNA library in a plasmid vector conMnued Synthesize the complementary strand Double stranded cDNA! Methylate (with an enzyme) the EcoRI sites (or whatever other sites you want to use) to protect them! Building a cDNA library in a plasmid vector conMnued Ligate a “linker” onto the ends of the double stranded cDNA Then, cleave with EcoRI to generate sMcky ends (for shuwling this cDNA piece into a vector for propagaMon Building a cDNA library in a plasmid vector conMnued These colonies are your collecMon of plasmid clones: your cDNA library! Recombinant DNA •  Overview and geneMc concepts •  Cloning –  Tools •  RestricMon endonucleases •  Ligase •  Polylnkers (MCS) –  Building a clone •  Libraries –  A source of clones –  Screening •  Defining what you’ve cloned •  PCR + expression cloning Next class! ...
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