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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