1. Recombination
May 2, 2018

2. Homologous Recombination
Breaks arising during DNA Replication
Accurately Repairing double-stranded Breaks
Exchange of Genetic Information between 2 chromosomes
It only takes places between DNA duplexes that have extensive regions of sequence similarity (homology)

3. HR Requires Sequence Homology

4. HR in DNA Replication Nick in a single strand
Replication Fork reaches the nick
Strands separate
Exonuclease degrades 5’ end

5. HR in DNA Replication Strand Invasion Strand Breakage DNA synthesis
Replication fork Re-established

6. HR in DNA Replication

7. Ionizing Radiation Ionizing radiation induced double-stranded breaks
X-rays
From Slide-Player
Ionizing –remove electrons

8. Double-stranded Breaks
dsDNA breaks are caused:
Directly, by ionizing radiation or other DNA damaging reagents
If unrepaired, they can result in Chromosomal Abnormalities

11. Homologous Recombination
Homologous recombination is one mechanism used to repair double-stranded DNA breaks
DNA repair proteins are recruited to the double-stranded break
3’ overhangs are created
Rad51 bings to the single stranded overhangs
Promotes “strand-invasion” on a the “homologous” chromosome.
The strand-invasion creates new sites for DNA synthesis
After DNA synthesis there are junctions that must be repaired.

12. Figure 11-4a 12

13. Generation of 3’ Overhangs
Figure 11-4b 13

14. Strand Invasion
Figure 11-4c 14

15. New Regions of DNA synthesis
Figure 11-4d 15

16. Resolution
Figure 4-11e 16

17. Homologous Recombination
RecA/RAD51 promotes “strand invasion”

18. Homologous Recombination
ssDNA is generated from the double-stranded break
RecA/RAD51 promotes “strand invasion”
Strand invasion creates new primer::template junctions that can be synthesized with DNA polymerase

19. RecA/Rad51 proteins RecA/Rad51 proteins form long filaments
Box
RecA/Rad51 proteins form long filaments
Higher-order nuclear structures in Rad51-overexpressing cells. (A) Immunofluorescence staining of growth-arrested TGR cells reveals discrete nuclear foci and higher-order structures of overexpressed Rad51 protein (green) in a high percentage of cells. Nuclei are counterstained with DAPI (blue). Bar, 10 μm. (B) Ultrathin cross (left-hand side) and longitudinal sections (right) of nuclear Rad51 structures in TGR nuclei after pre-embedding immunolabeling with anti-Rad51 antibodies and 12 nm colloidal gold. Bar, 100 nm
RecA/Rad51 proteins have a role in the invasion process
Raderschall E et al. J Cell Sci 2002;115: 19

20. How were Rad proteins discovered?
You guessed it! E. coli
Box
RecA protein was first identified in E. coli
DNA is integrated in host chromosomes by homologous recombination is required 20

21. RecA/Rad51 proteins RecA protein was first identified in E. coli
Box
RecA protein was first identified in E. coli
Question: Could mutations in genes that are required for recombination be identified.
Scientists made a Hfr cell that would donate Ade+ genes.
Donor (Hfr): Ade+; Leu -
Requires leucine supplementation
Recipient (F-): Leu+; Ade-
Requires adenine supplementation 21

22. If a recombination event occurs bacteria can grow without any
RecA/Rad51 proteins
Box
RecA protein was first identified in E. coli
Question: Could mutations in genes that are required for recombination be identified.
Scientists made a Hfr cell that would donate Ade+ genes.
Donor (Hfr): Ade+; Leu -
Requires leucine supplementation
Recipient (F-): Leu+; Ade-
Requires adenine supplementation
If a recombination event occurs bacteria can grow without any
supplementation
If recombination occurred, what would be the phenotype of the recipient cell. 22

23. RecA/Rad51 proteins RecA protein was first identified in E. coli
Box
RecA protein was first identified in E. coli
Question: Could mutations in genes that are required for recombination be identified.
Scientists made a Hfr cell that would donate Ade+ genes.
Donor (Hfr): Ade+; Leu -
Requires leucine supplementation
Recipient (F-): Leu+; Ade-
Requires adenine supplementation
Randomly mutagenized the recipient cell and asked if recombination was inhibited. 23

24. RecA/Rad51 proteins discovery
Box
Screened by then 2,000 mutagenize strains!
Recombination event:
Leu+, Ade+
Plated both the Donor (Hfr) and Recipient Strains on plates
with supplementation
Replica plated on plates without supplementation 24

25. RecA/Rad51 proteins discovery
Box
Expected: Recombination will allow the bacteria to grow on the plates without supplementation
Mutants: If bacteria have a mutation that prevents recombination, then they will not be able to grow on the plates without supplementation
Which bacteria clones might have this type of mutation?
Screened by then 2,000 mutagenize strains!
Recombination event:
Leu+, Ade+
Plated both the Donor (Hfr) and Recipient Strains on plates
with supplementation
Replica plated on plates without supplementation 25

26. RecA/Rad51 proteins discovery
Box
Expected: Recombination will allow the bacteria to grow on the plates without supplement
Mutants: If bacteria have a mutation that prevents recombination, then they will not be able to grow on the plates without supplementation.
Which bacteria clones might have this type of mutation?
Screened by then 2,000 mutagenize strains!
Recombination event:
Leu+, Ade+
Plated both the Donor (Hfr) and Recipient Strains on plates
with supplementation
Replica plated on plates without supplementation 26

27. RecA/Rad51 proteins discovery
Box
Screened by then 2,000 mutagenize strains!
Recombination event:
Leu+, Ade+
Plated both the Hfr and Recipient Strains on plates with supplemented
Replica plated on media without supplementation 27

28. Box
RecA/Rad51 proteins
RecA monomer has more than one DNA-binding site so it can bind either ssDNA or dsDNA
-It helps to catalyze strand invasion and hyrbridization 28