DNA STRUCTURE & REPLICATION

1. DNA STRUCTURE & REPLICATION

2. Last class... • We walked through the historical time line of the discovery of DNA • We ended with James Watson, Francis Crick, and Rosalind Franklin • Franklin was the first to capture an image of DNA • But it was Watson and Crick who finally discovered the true structure

3. Watson and Crick’s structure • Double helix that is comparable to a ladder. • The uprights are made of: • DEOXYRIBOSE sugars connected by PHOSPHATE groups • Held together by phosphodiester linkages • The ladder rungs are made of: • NITROGENOUS bases

4. A Closer Look... Phosphate • Same as you have seen before • 1 phosphorus atom • 4 oxygen atoms • Overall –ve charge • DNA is –ve!!! Deoxyribose Sugar • 5 carbons • 2’ C is missing an oxygen • 3’ C and 5’ C are most important

5. A Closer Look... Adenine Thymine Guanine Cytosine

6. A Closer Look... Adenine Thymine Guanine Cytosine PURINES The AGgies are PURe. PYRAMIDINES

7. A Closer Look... • A purine will always bond with a pyramidine • How nucleotide bases bond together: • A & T share 2 H-bonds C & G share 3 H-bonds • **The H-bonding gives DNA its stability!

8. A Closer Look... *** Thymine can not bond with Guanine due to lack of H-bonding*** *** Adenine can not bond with Cytosine due to lack of H-bonding***

9. Antiparallel... • DNA is said to run ANTI-PARALLEL • One strand runs in the 5’  3’ direction • The other runs in the 3’  5’ direction • The 5’ refers to the 5th carbon on the deoxyribose sugar • The 3’ refers to the 3rd carbon on the deoxyribose sugar • The 5’ end terminates with an phosphate group • The 3’ end terminates with an -OH group

10. Antiparallel...

11. DNA REPLICATION The Basics: • Cells can reproduce (as you have seen in mitosis) • Genetic information is divided equally from parent cell into daughter cells • Identical genetic information from parent to daughter cell is important to maintain identical cellular function • From their structure, Watson and Crick could tell: • H-bonds between nucleotides could break • DNA could ‘unzip’ • Each strand could act as a template to build a complementary strand

12. DNA REPLICATION Meselson & Stahl – important experiment • In 1958, suggested that DNA replication is SEMI-CONSERVATIVE • Each daughter cell receives one strand of parental DNA • Conservative = one daughter receives both strand

13. DNA REPLICATION Procedure • Grew E. Coli in a nutrient rich medium in 15N isotope • Allowed to replicate 17 times • thus all DNA should contain 15N • Next, bacteria with 15N were transferred to a medium of 14N • Now, 14N should be found in daughter DNA • One strand should be heavier than the other • As replication continues, more 14N should be found • All samples were centrifuged to separate by density • Results....

14. DNA REPLICATION Results • Tube A = DNA of cells before switching to 14N • Tube B = DNA after 1st replication of 15N in 14N sol’n • Both 14N and 15N • Tube C = DNA after 2nd replication • Intermediate band (14N+15N) and a light band (14N only) Tube A Tube B Tube C

15. DNA REPLICATION Conclusion • Original strands are still present after many replications, thus semi-conversative • They must act as a template • NOT conservative or we would see one heavy band and one light band Tube A Tube B Tube C

16. DNA REPLICATION The Roster • Many enzymes are used in this process • DNA helicase • DNA gyrase • DNA polymerase III • DNA polymerase I • DNA ligase • RNA primase • All have a very specific function in this process *Proteins can’t exist without DNA, but DNA has no function without proteins*

17. DNA REPLICATION The Process 1. DNA Helicase unwinds DNA by breaking H-bonds • Obviously, they are going to want to rebound (anneal) • Luckily, single-stranded binding proteins (SSBs) bind to exposed DNA blocking H-bonding 2. DNA Gyrasehelps the unwinding process by relieving any excess tension • DNA can’t be unwound all at once as it is too big. • The length of DNA in 1 chromosome is 1cm • The diameter of a cell 0.00005cm!! • To avoid this we need to replicate in regions

18. DNA REPLICATION

19. DNA REPLICATION The Process 3. DNA replication proceeds in the direction of the replication fork • Replication bubbles occur when two replication forks are close to one another 4. DNA polymerase III starts to build complementary strand

20. DNA REPLICATION The Process • Before DNA polymerase III can initiate a new strand by itself • It needs a PRIMER 5. RNA PRIMASE allows an RNA primerto be added • RNA primer marks the startof the initiation sequence

21. DNA REPLICATION The Process 6. Elongation - DNA polymerase: • Synthesizes DNA from 5’  3’ direction • Adds free nucleotides to the 3’ end • Elongation occurs easily on the 3’  5’ strand • LEADING STRAND • The 5’  3’ strandcreates short fragments • LAGGING STRAND

22. DNA REPLICATION The Process • RNA primers must be continually added to the 5’3’ parent strand to create lagging strand • This allows DNA polymerase III to build short fragments called OKAZAKI FRAGMENTS

23. DNA REPLICATION The Process 7. DNA polymerase I • Removes RNA primers • Replaces them withappropriate nucleotides 8. DNA Ligase • Joins one Okazaki fragment to another • Creates a phosphodiester bond

24. DNA REPLICATION The Process 9. When mistakes occur, DNA polymerase III & DNA polymerase I act as exonucleases • They can backtrack to remove the incorrect nucleotide and replace it with the correct one • A form of proof-reading

25. DNA REPLICATION Recap