1. DNA replication Sections 8.3 -8.5 Biology 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. 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

4. 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

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. Eukaryotes vs. Prokaryotes 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

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. DNA Polymerase is 1-directional 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

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

15. 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.

16. 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

17. 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?

18. http://www.bioteach.ubc.ca/TeachingResources/MolecularBiology/DNAReplication.sw f www.johnkyrk.com