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Virginia Tech BCHM 4116 - Exam 2 Study Guide

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BCHM 4116 1st EditionExam # 2 Study Guide Lectures: 14 - 23Lecture 14 (Feb 20)DNA REPLICATIONWhat illustrates that DNA is the genetic material? Animal cloning by nuclear transfer shows that DNA is genetic material. The method used, animal cloning, is not designed to show that DNA is genetic material, but it shows that maybe DNA and proteins and other substances that go into cloning may be the genetic material. What is the DNA synthesis reaction?The incoming base is selected with DNA polymerase active site (WC basepair with template strand)Nucleophilic attack by 3’-OH on alpha-phosphate of incoming dNTPNew phosphoestr bond formed = chain growth in 5’ to 3’ direction (antiparallel to template strand)Release of PPISubsequent hydrolysis of PPi by inorganic pyrophosphatase renders effectively irreversible. What is the universal mechanism for all polymerases?Two metal ions on incoming nucleotide coordinate to phsophatesMetal ions interact with D705 and D882“A” promotes nucleophilic attach of 3’O in alpha-phosphate of dNTP“B” assists departure of pyrophosphateA and B help stabilize transition state on alpha-phosphate. What is DNA replication?DNA replication is semi-conservative. This means that one of the two original strands is conserved in each progeny molecule. This picture depicts the grey DNA molecule as the originals, and by following the grey strands through the diagram, you can see what semiconservative means.How is DNA replicated?DNA replication is bi-directional. A pocket is made that opens up the two strands, and replication starts at the ‘fork’. This is located at the ends of the bubble, as depicted in the diagram. The enzymes responsible for unraveling the double helices is called helicases. And a topoisomerase that helps overcome torsional stress by phosphodiester bond breakage and reunion is called DNA gyrase.How does DNA replication occur?DNA replication is semi-discontinuous. DNA replication occurs in the 5’ to 3’ direction only. So The two strands that are being replicated are named two different things. The leading strand is replicated continuously, however the lagging strand is replicated in segments. This occurs because replication can only occur in one direction. The fragments that make up the lagging strand are called okazaki fragments. What are the general requirements for DNA synthesis?1. Template2. Primer with 3’ OH (RNA)3. dNTPs4. DNA polymerase and accessory proteinsWhat are the major types of DNA polymerases and what are their functions?DNA polymerase 1: It has 3’ to 5’ exonuclease activity  proofreading ability5’ to 3’ exonuclease  removes RNA primers and fills in gaps on lagging strand Poly I is also involved in DNA repair and removal of RNA primer. DNA Polymerase IIIPol III is a complex replicative DNA polymeraseCore: alpha (polymerase), epsilon (3’-->5’exonuclease), and theta (epsilon stabilization)Auxiliary subunits of Pol III: increase activity and processivityPol III holoenzyme is a dimer capable of the synthesis of the leading strand and the precursors ofthe lagging strandWhat are the enzymes involved in DNA replication?Gyrase: topoisomerase II negative supercoilingHelicase: unwinds DNAssDNA is coated with SSB (ss DNA-binding protein)Primase: periodically primes synthesis on the lagging strandEach half of the dimeric replicative polymerase (Pol III) is a ‘core’ polymerase bound to its template strand by a beta-subunit sliding clampDNA polymerase I and DNA ligase act on the lagging strand to remove RNA primers, replace them with DNA, and ligate the Okazaki fragments What are the stages of DNA replication in E. coli?InitiationoriC for E. coli, a 245 bp DNA sequenceoriC for E. coli, a 245 bp DNA sequenceDnaA (protein): binds to 4 DnaA boxes (DNA)DnaB: helicase activity unwinds DNA (assisted by DNA gyrase) SSB: coats unwound ssDNAHU (histone like DNA binding protein): prevents non-specific binding of DnaA Elongation by Pol III holoenzyme TerminationTerminus region (Ter)Tus protein bound to Ter stops DnaB helicaseDecatenation by DNA topoisomeraseWhat is the replication factory?A replication factory “fixed” to a cellular substructure extrudes loops of newly synthesized DNA s parental DNA duplex is fed in from the sides. Parental DNA strands are green; newly synthesized strands are blue; small circles indicate origins of replications.Lecture 15 (Feb 23)DNA REPLICATION (TELOMERE; RETROVIRUS)How is DNA replication in eukaryotic?Replication is initiated at multiple origins. DNA synthesis occurs during S phase. Replication occurs once per cell cycle. How is it regulated?Checkpoint: molecular controls (usually protiens) that control entry into the next stage. Regulation: cyclin-dependent protein kinase triggers DNA replication How are the ends of chromosomes replicated?When the ends of chromosomes are replicated, the lagging strand has a primer at the end. This primer, once it’s taken off nucleotides cannot be added to the end because to add nuclotides you need a primer, and since its at the end of the chromosome there is no place to attach a primer (Figure a). To overcome this problem, telomerase attaches to the bottom strand and elongates the top strand. The once telomerase comes off, polymerase can come and extend the bottom strand because (Figure b).Telomerase: Ribonucleoprotein complex responsible for maintaining telomere: 1. RNA serves as template2. RNA-dependent DNA polymerase3. Use 3’ end of the DNA as primerWhat is reverse transcriptase?Reverse transcriptase (RT) is an RNA dependent DNA polymeraseHIV virus uses this. Lecture 16 (Feb 25)DNA RECOMBINATIONWhat are the types of recombination?Homologous recombination, site specific, transposition, illegitimate. What is the Holliday model?The holiday model explains how homologous recombination works. A step by step viewing of the model and following the colors is the best way to learn how recombination.Here is a good diagram. A different perspectiveWhat are the proteins involved in this and what do they do?1. Ss nicks = RecBCD2. Strand invation = RecA3. RecA = recombinase4. Branch migration = RuvAB5. Junction resolution (RuvC)Lecture 17 (Feb 27)DNA RECOMBINATIONWhat is site specific recombination?Cre/loxP site-specific recombination: Cre is a recombinase from bacteriophaste Pi which mediate recombination between specific loxP sites (2x13 bp inverted repeat and a 8 bp spacer)What are transposable elements?Transposable elements are segments of DNA


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