1. DNA Recombination Roles Types Homologous recombination in E.coli
Transposable elements

2. Biological Roles for Recombination
Generating new gene/allele combinations (crossing over during meiosis)
Generating new genes (e.g., Immuno- globulin rearrangement)
Integration of a specific DNA element (or virus)
DNA repair

3. Practical Uses of Recombination
Used to map genes on chromosomes
- recombination frequency proportional to distance between genes
2. Making transgenic cells and organisms

4. Map of Chromosome I of Chlamydomonas reinhardtii
Top map is a map of molecular markers, bottom one is a map of phenotypic markers on the same chromosome
cM = centiMorgan; unit of recombination frequency
1 cM = 1% recombination frequency
Chlamydomonas Genetics Center

5. Types of Recombination
Homologous - occurs between sequences that are nearly identical (e.g., during meiosis)
Site-Specific - occurs between sequences with a limited stretch of similarity; involves specific sites
Transposition – DNA element moves from one site to another, usually little sequence similarity involved

6. Examples of (mostly) Homologous Recombination
Fig. 22.1
Crossovers involving circular chromosomes can give different outcomes than the linear DNAs in (a).

7. Holliday Model
R. Holliday (1964)
Holliday Junctions form during recombination
HJs can be resolved 2 ways, only one produces true recombinant molecules
Fig in 2nd edition
patch

8. EM of a Holliday Junction w/a few melted base pairs around junction
Fig. 22.3

9. The recBCD Pathway of Homologous Recombination
Part I: Nicking and Exchanging
From 1st or 2nd edition, Fig. in 3rd edition slightly different
Fig a-d

10. recBCD Pathway of Homologous Recomb. Part I: Nicking and Exchanging
A nick is created in one strand by recBCD at a Chi sequence (GCTGGTGG), found every 5000 bp.
Unwinding of DNA containing Chi sequence by recBCD allows binding of SSB and recA.
recA promotes strand invasion into homologous DNA, displacing one strand.
The displaced strand base-pairs with the single strand left behind on the other chromosome.
The displaced and now paired strand is nicked (by recBCD?) to complete strand exchange.

11. recBCD Pathway of Homologous Recombination
Part II: Branch Migration and Resolution
Nicks are sealed by DNA ligase.
Fig f-h

12. recBCD Pathway of Homologous Recom
recBCD Pathway of Homologous Recom. Part II: Branch Migration and Resolution
Nicks are sealed  Holliday Junction
Branch migration (ruvA + ruvB)
Resolution of Holliday Junction (ruvC)

13. RecBCD : A Complex Enzyme
RecBCD has:
Endonuclease subunits (recBC) that cut one DNA strand close to Chi sequence.
DNA helicase activity (recD subunit) and
a DNA-dependent ATPase activity
unwinds DNA to generate the 3’ SS tails

14. RecA 38 kDa protein that polymerizes onto SS DNA 5’-3’
Catalyzes strand exchange, also an ATPase
Also binds DS DNA, but not as strongly as SS
Homologues found in eukaryotes (Rad51, etc.), mitochondria, and chloroplasts.
Figure not in 3rd edition.

15. RecA binds preferentially to SS DNA and will catalyze invasion of a DS DNA molecule by a SS homologue.
Important for many types of homologous recombination, such as during meoisis (in yeast).
RecA is the bacterial gene, Rad51 is the nuclear homologue in eukaryotes.
Fig in Buchanan et al.

16. RecA Function Dissected
3 steps of strand exchange:
Pre-synapsis: recA coats single-stranded DNA (accelerated by SSB, so get more relaxed structure).
Synapsis: alignment of complementary sequences in SS and DS DNA (paranemic or side-by-side structure).
Post-synapsis or strand-exchange: SS DNA replaces the same strand in the duplex to form a new DS DNA (requires ATP hydrolysis).

17. RuvA and RuvB DNA helicase that catalyzes branch migration
RuvA tetramer binds to HJ (each DNA helix between subunits), forces it into square planar conformation
2 copies of RuvB bind at the HJ (to RuvA and 2 of the DNA helices)
RuvB is a hexamer ring, has helicase & ATPase activity
Branch migration is in the direction of recA mediated strand-exchange

18. RuvA/RuvB/DNA Complex
Shows RuvB encircling DNA duplexes

19. RuvB RuvA Similar to Figure 22.13 Model based on EM images.
RuvA removed for visual purposes only
Similar to Figure Model based on EM images.

20. RuvC : resolvase Endonuclease that cuts 2 strands of HJ
Binds to HJ as a dimer (that already has RuvA/RuvB)
Consensus sequence: (A/T)TT (G/C)
- occurs frequently in E. coli genome
- branch migration needed to reach consensus sequence!

21. RuvC bound to a HJ
The formation of the dimer in this way positions the active sites so they can cut the 2 strands shown. The yellow balls in figure b are the phosphates on each strand that could be attacked by RuvC.
Fig

22. A model for binding of RuvA, RuvB, and RuvC to a HJ.
Fig b

23. Meiotic Recomb. in Yeast - is initiated by a double-strand break (DSB)
Spo11 makes the Double-strand break
Fig

24. Repair of double-strand breaks (DSBs) in non-dividing or mitotic cells
DSBs probably most severe form of DNA damage, can cause loss of genes or even cell death (apoptosis)
DSBs caused by:
- ionizing radiation
- certain chemicals
- some enzymes (topoisomerases, endonucleases)
- torsional stress

25. 2 general ways to repair DSBs:
Homologous recombination (HR) - repair of broken DNA using the intact homologue, very similar to meiotic recombination. Very accurate.
Non-homologous end joining (NHEJ) - ligating non-homologous ends. Prone to errors, ends can be damaged before religation (genetic material lost) or get translocations. (Mechanism in Fig 20.38)
Usage: NHEJ >> HR in plants and animals