Today’s Outline

1. Today’s Outline • Study Notes 6b Due! • Take up exam • View exam on Friday between 2-4pm in C-4. • Written appeals-process • DNA Replication • Replication • PCR • Telomeres • DNA Replication Tutorial 3-5pm Friday

2. Write Down: What is one thing you learned about DNA replication from the readings?

4. To Understand DNA Replication, Understand the Parts • Helicase • Topoisomerase • Single Strand Binding Proteins • DNA Polymerase III • Sliding Clamp • DNA Primase

5. Helicase Helicase unzips DNA.

6. Topoisomerase Relieves the tension produced when DNA is unzipped.

7. Single Strand Binding Proteins Stabilizes DNA and prevents it from binding to parallel strand.

8. DNA Polymerase III Adds dNTPs to the newly synthesized strand on the leading strand. Adds in a 5’  3’ direction.

9. Sliding Clamp Holds DNA Polymerase III in place.

10. DNA Primase Nucleotides can ONLY be added to the 3’ end of an already-attached nucleotide. A primer provides this. DNA Primase builds the primer.

11. DNA

12. Polymerase Chain Reaction • What does polymerase do? • What if…. • I put an entire genome in a test tube. • I added DNA polymerase III and dNTPs. • I added very specific DNA primers that mapped onto a specific gene. • Then I started heating and cooling the DNA over and over and over and over….

13. Polymerase Chain Reaction 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ • taq polymerase • dNTPs • primers • buffer

14. Discuss: • Find three similarities and three differences between (i) DNA replication on the leading strand and (ii) DNA replication via PCR. (Debrief using name cards)

16. The 1st Challenge of Lagging Strand Replication • DNA Primaseis used over and over • DNA Primase lays down ribonucleic acid. Solution: an enzyme that can replace RNA nucleotides with DNA nucleotides  DNA Pol1

18. The 2nd Challenge of Lagging Strand Replication Solution: an enzyme that can connect 3’ carbons to phosphate groups  LIGASE

20. 5 Ends of parental DNA strands Leading strand Lagging strand 3 5 3 Last fragment Previous fragment Lagging strand RNA primer 5 3 Parental strand Removal of primers and replacement with DNA where a 3 end is available DNA Pol1 Can only add nucleotides to the 3’ end of existing DNA. 5 3 Telomeres protect an organism’s genes from being eroded during successive rounds of DNA replication. In humans the sequence TTAGGG is repeated between 100 and 1000 times. Second round of replication 5 New leading strand 3 5 New lagging strand 3 Further rounds of replication Shorter and shorter daughter molecules

21. Which of these replaces RNA with DNA? • DNA Polymerase 1 • DNA Polymerase 3 • DNA Primase • Ligase • Nuclease

22. Which of these glues 3’ and 5’ DNA ends together? • Ligase • DNA Polymerase 1 • Binding Proteins • DNA Polymerase 3 • DNA Primase

23. Which of these adds new nucleotides to the leading strand? • DNA Polymerase 3 • The Sliding Clamp • DNA Polymerase 1 • DNA Primase • Binding Proteins

24. Which of these is not needed by the leading strand: • DNA Polymerase 1 • Binding Proteins • DNA Primase • The Sliding Clamp • DNA Polymerase 3

25. Which of these is not needed by the lagging strand: • The Sliding Clamp • DNA Polymerase 1 • Binding Proteins • DNA Polymerase 3 • All are needed

26. Why are telomeres needed? • Primers can’t bind to 5’ ends of DNA • DNA polymerase 1 can’t add nucleotides to 5’ ends of DNA • Okazaki fragments don’t form at the end of DNA strands • Helicase can’t bind to the end of DNA strands. • None of the above.

27. Write Down: • On a scale of 1 to 10, how well do you understand DNA replication? • What is one thing you understand really well? • What is one thing you do not understand?

28. Coming Up: • Sanger Sequencing (Tues) • Molecular Sculpting of DNA replication (Tues) • Bonus assignment due (see Lecture Schedule) • Field trip sign up • DNA Replication Tutorial 3-5pm Friday