BCHM 4116 1st Edition Exam 2 Study Guide Lectures 14 23 Lecture 14 Feb 20 DNA REPLICATION What 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 dNTP New phosphoestr bond formed chain growth in 5 to 3 direction antiparallel to template strand Release of PPI Subsequent 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 phsophates Metal ions interact with D705 and D882 A promotes nucleophilic attach of 3 O in alpha phosphate of dNTP B assists departure of pyrophosphate A 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 Template 2 Primer with 3 OH RNA 3 dNTPs 4 DNA polymerase and accessory proteins What are the major types of DNA polymerases and what are their functions DNA polymerase 1 It has 3 to 5 exonuclease activity proofreading ability 5 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 III Pol III is a complex replicative DNA polymerase Core alpha polymerase epsilon 3 5 exonuclease and theta epsilon stabilization Auxiliary subunits of Pol III increase activity and processivity Pol III holoenzyme is a dimer capable of the synthesis of the leading strand and the precursors of the lagging strand What are the enzymes involved in DNA replication Gyrase topoisomerase II negative supercoiling Helicase unwinds DNA ssDNA is coated with SSB ss DNA binding protein Primase periodically primes synthesis on the lagging strand Each half of the dimeric replicative polymerase Pol III is a core polymerase bound to its template strand by a beta subunit sliding clamp DNA 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 Initiation oriC for E coli a 245 bp DNA sequence oriC for E coli a 245 bp DNA sequence DnaA protein binds to 4 DnaA boxes DNA DnaB helicase activity unwinds DNA assisted by DNA gyrase SSB coats unwound ssDNA HU histone like DNA binding protein prevents non specific binding of DnaA Elongation by Pol III holoenzyme Termination Terminus region Ter Tus protein bound to Ter stops DnaB helicase Decatenation by DNA topoisomerase What 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 template 2 RNA dependent DNA polymerase 3 Use 3 end of the DNA as primer What is reverse transcriptase Reverse transcriptase RT is an RNA dependent DNA polymerase HIV virus uses this Lecture 16 Feb 25 DNA RECOMBINATION What 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 perspective What are the proteins involved in this and what do they do 1 Ss nicks RecBCD 2 Strand invation RecA 3 RecA recombinase 4 Branch migration RuvAB 5 Junction resolution RuvC Lecture 17 Feb 27 DNA RECOMBINATION What 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 that jump around the genome Lecture 18 Mar 2 DNA RECOMBINATION AND ITS APPLICATIONS DNA recombination can be used for gene therapy Applications exist so that defective genes or