BIMM 100 Lecture 4

BIMM 100 Lecture 4 - BIMM100 Lecture 4 Key molecular...

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Unformatted text preview: BIMM100 Lecture 4 Key molecular processes III & IV Read: Lodish 145 ­160 •  Chapter 4 –  Finished: •  Nucleic acids •  TranscripEon •  TranslaEon –  ConEnuing: Remember to read over experiment 4 ­29 Also, remember to review “key terms,” “review the concepts,” and “analyze the data” at the end of the chapter •  ReplicaEon •  Damage and repair •  Viruses •  Next week: Chapter 5 –  Review pages 165 ­176 Lecture summary •  DNA replicaEon –  –  –  –  The process The players CoordinaEon and bi ­direcEonal replicaEon Finishing •  DNA mistakes, damage, and repair –  Polymerase ­ errors and proofreading –  Damage and repair •  •  •  •  DeaminaEon Base excision Mismatch repair UV damage and nucleoEde excision repair –  Global vs. transcripEon coupled •  Non homologous end joining ­ the last chance •  Viruses DNA synthesis: dNTP polymerizaEon ComplicaEons of DNA replicaEon •  Arise from two properEes of DNA –  The two complementary strands are anEparallel –  DNA polymerases can only add nucleoEdes in a 5’ ­3’ direcEon •  DNA must be –  Unwound (helicases) –  Primed –  Ligated back together •  Specific enzymes perform these acEviEes DNA replicaEon •  Beginning the process –  The site: replicaEon origin •  Sequences are A ­T rich •  Size and sequence is variable –  Helicases •  Unwind DNA at the origin –  Primase (a special RNA polymerase) •  Forms a short, complimentary RNA primer on both DNA strands •  ElongaEon by DNA polymerase –  Site of acEon: replicaEon fork DNA replicaEon: solving the polarity problem! Reiji Okazaki (with his wife) made this discovery in 1968. He died seven years later, due to leukemia. He has sustained severe irradiaEon during the bombing of Hiroshima in 1945. Leading strand: conEnuous synthesis Lagging strand: disconEnuous ReplicaEon occurs bi ­direcEonally from each origin! DNA replicaEon: model of the SV40 replicaEon fork; the players SV40: simian virus 40 Normal and asymptomaEc in Rhesus monkeys ­ in immunodeficient animals, it causes kidney disease and demyelinaEon. Aher it’s discovery, it was detected in polio vaccine lots made between 1955 ­1961 (in the US). Soviet stocks of virus tested posiEve for the virus up to 1980! Luckily, it has never been 3ed to human disease… Primase ­ special RNA polymerase that forms short RNA primer Polα ­ DNA polymerase that extends primers Polδ ­ DNA elongaEng polymerase ­ has proofreading mechanism Other players: •  topoisomerase I: relieves torsional stress •  Rfc (replicaEon factor c) & PCNA (proliferaEng cell nuclear anEgen) •  Ribonuclease H & FEN1 •  DNA ligase: joins fragments •  RPA (replicaEon protein A) DNA replicaEon: the process (leading) 1.  Large T anEgen (from SV40) unwinds the parental DNA. Primase synthesizes a short RNA primer, which is extended by Polα. 2.  Single stranded regions of the template are bound by mulEple copies of the heterotrimeric protein (RPA) that keeps the template in an opEmal configuraEon for replicaEon. 3.  A complex of Polδ, Rfc, and PCNA synthesize DNA (it passes through a “hole” created by that complex). DNA replicaEon: PCNA surrounds the DNA Prevents the Polδ, Rfc, and PCNA complex from dissociaEng. DNA replicaEon: the process (lagging) 4.  Primers for lagging strand synthesis are synthesized by primase and Polα. 5.  3’ end of each primer synthesized is bound by Polδ, Rfc, and PCNA complex, which synthesizes most of the Okazaki fragment. DNA replicaEon: the process (ligaEon) •  Ribonuclease H and FEN I remove nucleoEdes at the 5’ ends of Okazaki fragments •  These are replaced by dNTPs added by Polδ as it extends the fragments •  Fragments are coupled by DNA ligase by phosphodiester bonds. •  At the ends of the DNA molecule, lagging strands cannot be replaced by dNTPs. This is achieved with telomerase. Polε also involved ­ unknown role CoordinaEng lagging and leading strand synthesis ­animaEon SV40 DNA replicaEon is bi ­direcEonal Experiment was performed to ask “is replicaEon uni ­or bi ­direcEonal?” “Is there one fork? Or two forks?” To perform this experiment, the circular viral chromosome was cut with an enzyme. This is now a “landmark” on the DNA. Data: the measurement from the center of the “bubbles” to the “landmark” was measured. ObservaEon: the centers were always a constant distance from the DNA ends. Conclusion: Two forks are present, replicaEng DNA in two different direcEons, from a common origin in the middle of the “bubble”. Bi ­direcEonal replicaEon: the way replicaEon normally happens… Two large T anEgen complexes bind at the origin. 1. Helicases move in opposite direcEons, unwinding DNA, making single strands that are bound to RPA proteins (energy from ATP hydrolysis). 2. Polα makes short primers (red) that are base ­paired to the two parental strands. 3. Polδ, PCNA, Rfc complexes replace the Polα and extend the short primers (make two leading strands (green) at each fork) 4. Helicases further unwind the parental DNA, and RPA proteins bind to the new single strands Bi ­direcEonal replicaEon 5. Polδ, PCNA, Rfc complexes keep extending the leading fragments. 6. Polα complexes synthesize primers for lagging strand synthesis at each replicaEon fork 7. Polδ, PCNA, Rfc complexes displace Polα and extend the Okazaki sequences (light green) Okazaki sequences are ligated to the 5’ ends of the leading strands. ReplicaEon conEnues by repeaEng these steps! Review: Bi ­direcEonal DNA replicaEon Lecture summary •  DNA replicaEon –  –  –  –  The process The players CoordinaEon and bi ­direcEonal replicaEon Finishing •  DNA mistakes, damage, and repair –  Polymerase ­ errors and proofreading –  Damage and repair •  •  •  •  DeaminaEon Base excision Mismatch repair UV damage and nucleoEde excision repair –  Global vs. transcripEon coupled •  Non homologous end joining ­ the last chance •  Viruses DNA mistakes, damage, and repair •  In a perfect world, replicaEon would proceed with no errors and DNA would be stable and would never be damaged •  Unfortunately, this is not the case: –  Mistakes are made in replicaEon, and DNA is damaged •  Fortunately, cells have compensatory and proofreading mechanisms to correct this. –  Key to genomic stability and integrity of geneEc informaEon –  Key to responding to environmental, chemical, and radiological insults •  How? Stay tuned… DefiniEons and clarificaEons •  Damage doesn’t necessarily lead to mutaEons! •  DNA can be damaged in a number of ways. –  Cleavage of chemical bonds in DNA –  Environmental agents (UV or ionizing radiaEon) –  Genotoxic chemicals •  From environment or your own cells! –  Copying errors in DNA polymerases •  If there is severe structural damage to the DNA, cells can die! DefiniEons and clarificaEons •  Some forms of damage can be recognized and repaired by a number of specialized cellular pathways that restore the original DNA sequence •  Some repair mechanisms are error ­prone –  They save the cell/organism from death, but can permanently change the geneEc sequence of the organism (a mutaEon). Bad news ­ even DNA polymerase makes mistakes… Well, only 1 out of 104 Emes… But, the overall mistake rate is only 1 out of 109. How can that be? When an incorrect base is incorporated, the base ­pairing between the 3’ nucleoEde and its complement doesn’t happen. Polymerase pauses, transfers the 3’ end of the chain to the exonuclease site, and the incorrect base is removed. 3’ end is transferred back to the polymerase site, and the region is copied correctly Why does this maner? •  DNA is exposed to 104 ­106 damaging events per day! •  Many human diseases are associated with mutaEons involved in DNA ­repair defects If unrepaired, deaminaEon leads to point mutaEons One of the most common causes of point mutaEons! MutaEon can no longer be recognized as “wrong.” Base excision (to repair T ­G mismatches and damaged bases) ­happens before replicaEon! Problem: which strand is normal? Which strand is mutant? Since the deaminaEon 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, effecEvely “cupng” 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 (occurs aher replicaEon) 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. MutaEons in these genes are Eed to hereditary nonpolyposis colorectal cancer! UV damage causes a specific type of DNA damage: pyrimidine dimers Recognized because they distort the normal shape of DNA. Causes problems in replicaEon and transcripEon! Discovered by studying cultured cells from paEents with xeroderma pigmentosum, who are predisposed towards developing melanoma and squamous cell carcinomas when they are exposed to UV light. Why? They lack funcEonal XPA genes (A ­G), which are essenEal for repairing these “lesions.” How is this repaired? NucleoEde excision repair 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. How is this repaired? NucleoEde excision repair 3. XP ­G (now funcEons 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 nucleoEdes. 4. DNA polymerase fills the gap, and ligase seals the ends. VariaEon on the same process: transcripEon coupled repair 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! EssenEal, because this directs repair to criEcal regions (regions being transcribed) so mutaEons will not be generated! 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! NHEJ ­ not just for DNA repair! •  EssenEal for diversity of the immune system –  For B and T cell diversity: •  Joining of variable (V), diversity (D), and joining (J) regions to assemble the variable region of the B cell (or T cell) receptor. These receptors bind to anEgens expressed on the surface of cells, iniEaEng signaling cascades to clear infecEons. –  In V(D)J recombinaEon, hairpin ­capped double ­strand breaks are created by RAG nucleases (cleave DNA at specific sites). These hairpins are then joined by NHEJ. A specialized DNA polymerase called TDT (only expressed in lymph Essue), adds nontemplated nucleoEdes to the ends before the break is joined. –  Unlike typical cellular NHEJ, in which accurate repair is the most favorable outcome, error ­prone repair in V(D)J recombinaEon is beneficial! »  WHY? It maximizes diversity in the coding sequence of these genes. PaEents with mutaEons in NHEJ genes are unable to produce funcEonal B cells and T cells and suffer from severe combined immunodeficiency (SCID) ­ the “bubble boy” syndrome. SCID ­ not just caused by NHEJ mutaEons! •  Second most common cause? –  DefecEve adenosine deaminase (ADA), necessary for the breakdown of purines. •  Low ADA causes accumulaEon of dATP, which inhibits the acEvity of ribonucleoEde reductase, the enzyme that reduces ribonucleoEdes to generate deoxyribonucleoEdes. •  The effecEveness of the immune system depends upon lymphocyte proliferaEon (and dNTP synthesis). Without funcEonal ribonucleoEde reductase, lymphocyte proliferaEon is inhibited and the immune system is compromised. Homologous recombinaEon •  Repair of damaged sequence by a segment copied from the same (or highly homologous) DNA sequence on a homologous chromosome (in diploid organisms) or a sister chromaEd. •  Important! –  Several human cancers are caused by mutaEons in these pathways. •  Ex: breast cancer is Eed to mutaEons in BRCA1 and BRCA2, both important genes in the process. –  Generates geneEc diversity among individuals of a species by allowing the exchange of large regions of chromosomes between maternally ­ derived and paternally derived chromosomes during meiosis (the generaEon of germ cells). –  We’ll talk about this more in Chapter 5. Don’t stress about pages 150 ­153 for now! Lecture summary •  DNA replicaEon –  –  –  –  The process The players CoordinaEon and bi ­direcEonal replicaEon Finishing •  DNA mistakes, damage, and repair –  Polymerase ­ errors and proofreading –  Damage and repair •  •  •  •  DeaminaEon Base excision Mismatch repair UV damage and nucleoEde excision repair –  Global vs. transcripEon coupled •  Non homologous end joining ­ the last chance •  Viruses CHAPTER 4 is done! 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 •  MulEple mechanisms of replicaEon and propagaEon •  Important for human, animal, and plant health Growth and enumeraEon of viruses: the plaque assay Plaque: an absence of cells (i.e. presence of a virion) The lyEc 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, essenEal for packaging and degradaEon of the host cell. Eventually the cell dies, spewing virus to infect other cells Retroviruses: the life cycle Animated cycle of retroviral infecEon See two other animaEons, too! ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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