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UW-Madison BIOLOGY 151 - DNA Replication

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BIO 151 1nd Edition Lecture 16 Outline of Last Lecture - Erwin Chargaff’s rules- DNA structureo Double helixo DNA-DNA Hybridizationo Nucleic acidsOutline of Current Lecture - DNA Replication- DNA Replication enzymes - Leading/Lagging enzymes- Polymerase Chain Reaction- Replication accuracyo Fixing DNA defectsCurrent Lectureo Preparing for DNA Replication, NEED:o DNA templateo “Free” nucleotides to build new strandso Proteins to perform specific jobso Steps for DNA Replication:o Job 1: separate the 2 strands Helicase: comes in and unzips double stranded DNAo Job2: Topoisomerase: relieve strain on the template to prevent supercoiling (prevents super coil or breaking of DNA.  if not careful, DNA could be super coiled, creating a huge knoto Job 3: Single stranded binding proteins: prevent strands from rejoining These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Job 4: DNA polymerase: links nucleotides 5’ to 3’ to synthesize new strad Makes new strand 5’ to 3’ Locate template strand, builds 5’ to 3’ to make strand complementary to strand Bring in nucleotide  2 phosphates will be cleaned off  brings energy into system since takes lots of energy to go 3’ to 5’o If DNA can only copy 5’  3’, then how does it work at Replication fork?o New strands synthesized in opposite directions from replication fork Leading strand synthesized TOWARDS fork Lagging strands synthesized AWAY from forko Both strands synthesized simultaneouslyo Leading Strand Synthesis: o Hallmark: continuouso Problem: DNA polymerase can’t initiate DNA synthesiso Primase: builds a small RNA strand (“primer”) complementary to template for leading strando Using this primer, DNA polymerase adds nucleotides in a continuous mannero Primase build a small RNA strand (“primer”) complementary to template for leading strand Primase is actually a RNA polymerase! Primase adds RNA primer to take advantage of DNA The RNA primer gets the attention of DNA polymeraseo Lagging strand Synthesis: away from fork, discontinuouso Primase reads template strand  makes primero Polymerase makes fragmento DNA polymerase detaches (short): Okazaki fragments: short fragmentso Another batch of primase, DNA polymerase comes in and makes another Okazakai Fragmento DNA polymerase replaces RNA with DNAo DNA ligase forms bonds with DNA fragments Ligase: makes covalent bonds to create backbone 2 Okazaki fragments  Lagging away from fork; behind leading; discontinuous System needs RNA to get DNA polymerase’s attentiono Lagging is discontinuous because… Want to be time efficient (continuous with leading strand) How long does this take: polymerase can make 500/min in humans- Copying entire genome takes a few hourso Semi-conservative replication in a tube: Polymerase Chain Reaction (PCR)o Need: DNA, known sequence  need to add primers that are complementary Primer dNTPs free nucleotides  polymerase makes strands out of these DNA polymerase Thermo Cydero Polymerase Chain Rxn Denature (melt DNA strands apart) (temp down) Anneal: primer binds DNA Elongate-DNA polymerase synthesizes new strand (temp up) Repeat 1-3  somewhere between 30-50 timeso DNA polymerase is unable to join adjacent DNA fragments, so ligase has to do that Primary function of DNA polymerase is to DNA strand elongationo How accurate is replication?o DNA polymerase makes 1 error ever 100,000 nucleotideso 3’ to 5’ Exonuclease: intrinsic repair function that DNA polymerase has  “eyes on back of head”: realizes mistake  reverses (3’ to 5’) to removes incorrect nucleotide and then adds correct nucleotideo Fixing DNA defects before replicationo UV light damages covalent bonds between adjacent T nucleotides causes buckling in strand Nuclease: recognizes problem, cut on either side of bad stuff DNA Polymerase (fills in gap)DNA ligase (make covalent bonds on fixed section)o 100 “proofreading” proteins!  after proofreading, only 1 in 10 billion nucleotides are incorrect human genome = 3.2 billion nucleotides so, only 32 unfixed mistakes/genome replication- if every enzyme is working correctly otherwise number could be a lot higher 2% of DNA codes for


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