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DNA Replication - greinerudsd

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					DNA replication
Sections 8.3 -8.5
   Biology 391
    DNA Synthesis
•   Who? - all living organisms
•   What? - making an exact duplicate of DNA
•   When? - during S phase of interphase
•   Where? – nucleus (or cytoplasm)
•   How? – via several enzymes
    – semi-conservative
    – Denature, Anneal Primers, Extend
• Why? – So that we can grow (divide)
   DNA Structure
• Sugar-Phosphate
  backbone
• Nitrogenous Base
   – C always pairs with G
   – A always pairs with T
   – Hydrogen bonds connect



• Antiparallel
   – 5’ phosphate
   – 3’ hydroxyl
         Steps of DNA replication
1. DENATURE
  – Binding of enzymes to existing DNA
    •   At replication origins
    •   Replisome: DNA and associated bound proteins
  – Double helix is unwound
2. ANNEALING
  – 4-15 nucleotides of RNA added to ssDNA
3. EXTENSION
  – Synthesis of a new matching strand for each
    existing strand
  – Leading versus Lagging strands
             Enzymes Involved
• Helicase – separate initial DNA strands
  – SSBP – prevent reannealing of DNA strands
• Primase – lay down initial RNA primers so DNA
  pol III can work
• DNA polymerase I – removes RNA primers
  (exonuclease) and replaces with DNA (CC#1)
• DNA polymerase III – main replication enzyme
• DNA Ligase – “glue” together small DNA
  fragments, seal backbone
                 DENATURING:
              starting DNA replication
• Replication Origins
   – A-T rich; easiest to break Why?
   – DNA is opened by initiator proteins and helicase
   – Different in Euks/Proks
        Eukaryotes vs. Prokaryotes
• Linear chromosomes     • Single circular
• Multiple replication     chromosome
  origins                • One origin of replication
• Synthesis occurs in    • Synthesis occurs in
  nucleus                  cytoplasm
• Only works in one      • Bidirectional
  direction              • DNA pol adds ~1000
• DNA pol adds ~100        bases per second
  bases per second
                ANNEALING:
• DNA Pol III needs a double stranded starting
  point, but replication requires the DNA to
  become single-stranded:
  – Solution: Put down short, temporary starter
    sequences.
  – Primase lays down short RNA oligonucleotides so
    that DNA pol III can bind
  – RNA is later removed and replaced by DNA
    nucleotides (via DNA pol I)
                EXTENSION:
        adding complimentary bases
• DNA Polymerase III is major enzyme
• 50 nucleotides/second/replication fork
• Each strand acts as a template for making a new
  double strand of DNA
   – Each half (strand) of the original DNA is known as a parent
     strand
   – The new, complementary strand of DNA that matches is
     called the daughter strand
    DNA Polymerase is 1-directional
• LEADING STRAND           • LAGGING STRAND
• Made 5’  3’             • Made 5’  3’
  continuously               discontinuously
• Follows replisome as     • Short segments =
  unwound                    Okazaki fragments
• 1 RNA primer necessary   • Goes in opposite
  at start                   direction to replisome
                           • Uses several RNA
                             primers
                           • Short segments ligated
                             afterwards
   SEMI-CONSERVATIVE REPLICATION
• ½ old DNA coding for ½ new DNA is known as SEMI-
  CONSERVATIVE REPLICATION
• During replication: each old double strand will:
  1. Unzip into 2 single strands, which will:
  2. Code for a complementary strand (A-T; G-C)
  3. Which will attach with H+ bonds to form:
  4. 2 new double strands with:
  5. 1 new single strand and 1 old single strand each
       Semi-Conservative Replication
•   http://www.dnaftb.org/dnaftb/20/concept/index.html
FYI: Sun directly damages DNA by bonding
thymine bases side-by-side. This can create
confusion during replication. Polymerase has
difficulty reading this abnormality and might
skip it, match just one A or interpret as a big
purine.
                  DNA Repair
• DNA pol - error rate of 1 in 10,000 bases
  – However proofreading minimizes this to 1 in 10mil
• Mutation  any change in DNA
  – Causes: Genetic error, environmental factors
• Excision Repair – recognize mismatched bp,
  break sugar-phosphate backbone, remove.
  DNA pol replaces and ligase connects
                 Recap…
• What enzyme unzips the DNA helix?
• What enzyme reads and matches nucleotides
  to the parent DNA strand?
• When does DNA replication happen?
• What is a replisome?
• If the parent strand read: ATCGGGTCGT what
  would the compliment be?
http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.sw
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