1. DNA Replication & Repair

2. We already know… Replication means  copy
DNA is replicated during interphase of the cell cycle
Replicated DNA is separated during anaphase of mitosis & anaphase II of meiosis
Cell cannot pass the G2 checkpoint if the DNA has not replicated fully

3. Remember: One strand of DNA runs 5’  3’
The other strand runs 3’  5’
Complementary
Antiparallel
Two strands are held together by hydrogen bonds
H bonds are easily broken
Especially at sites rich in A-T base pairs (why?)

4. Semi-conservative replication
“New” DNA has one strand from the original DNA & one new strand

5. How do we know?
Read about Meselson & Stahl’s experiments on p in your textbook
Animation:

6. Each strand of DNA acts as a template to build the complementary strand
Template = pattern
New nucleotides are added to the template from 5’ 3’

7. Step 1 Separate the strands
DNA helicase (enzyme) binds to the replication origin site and breaks the H bonds

9. Separated strands might anneal (stick back together)
SSBs (single-stranded binding proteins)
Stick to the exposed strands so they cannot reform

10. Take a piece of string.
Use a pen or pencil to poke a hole in between the strands.
Now continue to separate the strands up the length of the string.
What happens?
Why?

11. DNA Gyrase Enzyme Loosens the tension in the separated strands
Cuts the DNA & allows it to reattach after it relaxes
Also helps to relax DNA & unwind it initially

12. Replication bubble Place where DNA replication takes place
New strands are synthesized on both sides

13. Many replication bubbles are formed to speed up the process

14. Step 2 Complementary strands are assembled DNA Polymerase III
Adds nucleotides to the template in the 5’ – 3’ direction
Actually adds “deoxyribonucleoside triphosphates” (just like a nucleotide but with 2 extra phosphate groups)

15. Deoxyribonucleoside triphosphate

16. A small segment of RNA is added as a “primer”
Each new nucleotide is added to the 3’ carbon of the previous nucleotide
A small segment of RNA is added as a “primer”
Gets the strand started
10 – 60 base pairs in length
Built by the enzyme primase
Temporary

17. DNA polymerase III breaks off the extra 2 phosphate groups
Dehydration synthesis
Provides energy to add the next nucleotide
Extra phosphates recycled by the cell

19. Remember… DNA strands are antiparallel
New nucleotides are only added in one direction
So…
Only 1 strand can be synthesized continuously

21. Continuous strand = leading strand
Synthesized from 5’  3’ on a 3’  5’ template
Built toward the replication fork
Discontinuous strand = lagging strand
Synthesized from 5’  3’ in short pieces on the opposite template
Built away from the replication fork

22. Lagging strand… cont’d
Primers are added for each segment as the replication fork opens further
Short segments are called Okazaki Fragments

23. Step 3 New DNA is finished DNA Polymerase I DNA ligase
Removes RNA primers (from both strands)
Replaces them with DNA nucleotides
DNA ligase
Creates phosphodiester bonds to connect the Okazaki fragments

24. Finished! 2 copies of DNA, each with 1 old strand, 1 new strand
Twist into double helix shape

25. Quality Control
DNA polymerase III & I check the strands as they are built
“proofread”
If there is a mistake, either enzyme can be a “exonuclease” & cut out the mistake

26. Summary: Enzymes involved in DNA replication
DNA helicase
DNA gyrase
DNA polymerase III
RNA Primase
DNA polymerase I
DNA ligase
Describe the role of each enzyme…

28. Homework Questions
Page 223: #2, 4, 5, 6
Page 230: #2-4, 6, 9-14