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DNA replication Sections 8.3 -8.5 Biology 391

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

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DNA replication Sections 8.3 -8.5 Biology 391

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  1. DNA replicationSections 8.3 -8.5Biology 391

  2. 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)

  3. Sugar-Phosphate backbone Nitrogenous Base C always pairs with G A always pairs with T Hydrogen bonds connect Antiparallel 5’ phosphate 3’ hydroxyl DNA Structure

  4. Steps of DNA replication • DENATURE • Binding of enzymes to existing DNA • At replication origins • Replisome: DNA and associated bound proteins • Double helix is unwound • ANNEALING • 4-15 nucleotides of RNA added to ssDNA • EXTENSION • Synthesis of a new matching strand for each existing strand • Leading versus Lagging strands

  5. 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

  6. 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

  7. Linear chromosomes Multiple replication origins Synthesis occurs in nucleus Only works in one direction DNA pol adds ~100 bases per second Single circular chromosome One origin of replication Synthesis occurs in cytoplasm Bidirectional DNA pol adds ~1000 bases per second Eukaryotes vs. Prokaryotes

  8. 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)

  9. 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

  10. LEADING STRAND Made 5’  3’ continuously Follows replisome as unwound 1 RNA primer necessary at start LAGGING STRAND Made 5’  3’ discontinuously Short segments = Okazaki fragments Goes in opposite direction to replisome Uses several RNA primers Short segments ligated afterwards DNA Polymerase is 1-directional

  11. 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

  12. Semi-Conservative Replication • http://www.dnaftb.org/dnaftb/20/concept/index.html

  13. 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.

  14. 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

  15. 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?

  16. http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swfwww.johnkyrk.comhttp://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.swfwww.johnkyrk.com

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