1. Mammalians homologs of RecA proteins are called Rad51– why is this important?
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Mutations in BRCA1/2 and Rad51 are associated with breast cancer
BRCA1/2 proteins are part of the DNA repair proteins that are recruited to double-stranded breaks. 1

2. Rad52 proteins Rad52 Functions:
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Rad52 proteins
Rad52 Functions:
-Displaces single-stranded binding proteins allowing the binding of Rad51
-Promotes the annealing of complementary single-strands 2

3. Localization of Repair Proteins
Repair proteins are usually made at high levels and dispersed throughout the nucleus
Cell partially treated by X-rays
A – DAPI
B – BudR incorporation
C – Mre11 Antibodies

4. HR Resolution
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Box 4

5. Holiday Junctions RuvA RuvB
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Holiday Junctions
RuvA
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RuvB
Cleavage Vertically or Horizontally by an endonuclease RuvC in E. coli, resolvase in eukaryotes 5

6. HR Resolution
Box
Holiday Junctions
Box 6

7. HR in Meiosis Spo11 cleaves both strands, similar to a topoisomerase
Mre11, specialized nuclease chews back the 5’ end leaving a 3’ overhang

8. HR in Meiosis
90% in humans %

9. HR in Meiosis Meiosis yields equal number of gametes 2 Blue and 2 Red
Meiosis doesn’t always yields equal numbers of gametes
3 Blue and 1 Red

10. HR in Meiosis
Heteroduplex – a region of DNA helix formed from 1 paternal and 1 maternal strand
Are they identical?

11. Mismatch Repair in HR Gene Conversion caused by Mismatch Repair
Can Result in Loss of Heterozygosity

12. Mismatch Repair in HR
Mismatch Repair prevents recombination between poorly matched DNA sequences

13. HR Movie

15. Double-stranded breaks
Double-stranded break are disastrous for the cell
dsDNA breaks are repaired by
Homologous recombination
Nonhomologous end joining (NHEJ)

16. Nonhomologous end joining (NHEJ)
NHEJ is messy and error prone
NHEJ is also very complicated!
Ku 70/80 are proteins that bind to the ends of the ds break
Overhangs are created
Ligase can seal the strands
In the processing step, sequences can often deleted.
Meiosis yields equal number of gametes
2 Blue and 2 Red
VDJ recombination
C is lost!
GTTGG is lost!
NHEJ repairs double-stranded breaks but results in mutations.

17. NHEJ Movie

18. Nonhomologous end joining (NHEJ) is used to make mutations in genes!
Why would researchers want to make mutations in genes?
To study loss-of-function phenotypes.
How do scientists make mutations?
VDJ recombination
Add mutagens like the 5-Bromouracil
Ionization radiation
CAS9/CRISPR
Transposons

19. Genome Editing

20. Next Generation of Genome Editing?
CRISPR-Cas9
-Cas9 protein
-guide RNA

21. CRISPR/CAS9 is a technique that induced double-stranded breaks at specific sequences
How is the double stranded break repaired?

22. CAS9/CRISPR: Genome Editing
Generate Mutations in specific genes using NHEJ repair
Gene Therapy: Replace “Bad” genes with “Good” genes using HR repair

23. CAS9/CRISPR: Genome Editing
Target RNA
Cas9 Protein *
TARGET DNA sequence

24. CAS9/CRISPR: Genome Editing
To get the CAS9/CRISPR system to work, you need to make cells produce the Cas9 enzyme and the target/guide small RNA.
What type of promoter would you use to drive the expression of Cas9 enzyme?
What type of promoter would you use to drive the expression of the guide RNA?
Target RNA
Cas9 Protein *
TARGET DNA sequence

25. Types of RNA polymerases transcribe different types of genes
Poly II
CAS 9 protein is under a Poly II promoter
Target/Guide RNA protein is under a Poly III promoter. * Finding Poly III core promoter sequences was a major challenge in this research!
Poly III
Target RNA
Cas9 Protein *
TARGET DNA sequence

26. CRISPR/Cas9 Technologies uses
both NHEJ and HR
If the double-stranded DNA break is repaired by NHEJ, mutations are induced in the gene you are interested in studying!

27. CRISPR/Cas9 Technologies uses
both NHEJ and HR
If the double-stranded DNA break is repaired by NHEJ, mutations are induced in the gene you are interested in studying!
If you add a “repair” template with homologous sequences, then homologous recombination is used to repair the double-stranded break. This is how gene editing occurs: “replacing the good gene with the bad gene”

28. Homologous Recombination