1. Homologous Recombination
MCB 7200 Group 1 Ashley Patton Yanyang Cuo Samantha Shaw

2. Homologous Recombination
Biological Relevance
Repairs double-stranded breaks
Chromosome segregation during meiosis I
Genetic diversity in gametes
Mediated by two important enzymes
Rad51 and Dmc1
Occurs during late-S phase, G2, M
Define:
Homologous recombination is responsible for repairing double stranded breaks during mitosis. In meiosis, HR is responsible for creating genetic diversity in gametes to produce progeny that is genetically different from the maternal and paternal DNA.
When: DSB mitosis, to generate genetic variations during meiosis
Where: nucleus: sister chromatids
When: late S-phase, G2, M
How:
Filippo et al Mechanism of Eukaryotic Homologous Recombination

3. Construction of P. falciparum expression vectors
Homologous recombination is a convenient cloning alternative when suitable restriction sites are not available.
The method allows for the replacement of entire genes or small regions within a single gene to form chimeric genes.
The clones can also be transformed into E. coli.

4. Homologous Recombination
Double Strand Break Repair
Synthesis-dependent strand annealing
Migrating D loop
No Holiday Junction formation
Mitosis/Meiosis
Single strand annealing
Repair of double strand break by deletion of repeated DNA sequences
Break-induced repair
Telomeres
Replication forks

5. Homologous Recombination Mechanism
Filippo et al Mechanism of Eukaryotic Homologous Recombination

6. Prokaryotic Recombination
RecBCD
Helicase activity unwinds DNA
Exonuclease activity degrades both single strands
At CHI site
3′ - 5′ Exonuclease activity is inhibited
5′ -3′ activity enhanced yielding a single-stranded 3′ end.
This end becomes coated with multiple RecA proteins, which catalyze strand invasion and formation of a Holliday structure
Lodish et al., Molecular Cell Biology, 4th Fig 12-32

7. Prokaryotic Recombination
RecA
RecA binds ssDNA in the presence of ATP
RecA aligns the ssDNA with its homologous dsDNA region
RecA inserts the ssDNA into target DNA sequence forming a heteroduplex Holiday- type structure
Branch Migration and Resolution by RuvA,B,C
Lodish et al., Molecular Cell Biology, 4th Fig 12-35

10. Filippo et al. 2008. Mechanism of Eukaryotic Homologous Recombination

11. Summary
Used for double strand break repair and genetic diversity in meiosis.
Different mechanisms within the Eukaryotic and Prokaryotic cells.
Proteins are different with each system by have similar functions.

12. Summary: Prokaryotic vs Eukaryotic
C. Wyman et al. /DNA Repair 3 (2004) 827–833

13. References
Fillipo et al. Mechanism of Eukaryotic Homologous Recombination Annual Rev. Biochem. 77:
Lodish et al. Molecular Cell Biology W. H. Freeman. 4th edition.
Oldenburg et al. Recombination-mediated PCR directed plasmid construction in vivo in yeast Nucleic Acids Research. Vol. 25, No. 2.
Wyman et al. Homologous recombination-mediated double-strand break repair. (2004) DNA Repair. 3:827–833.

14. Questions?