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

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. 2008. Mechanism of Eukaryotic Homologous Recombination

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.

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

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

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

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

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Filippo et al. 2008. Mechanism of Eukaryotic Homologous Recombination

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.

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

References Fillipo et al. Mechanism of Eukaryotic Homologous Recombination. 2008. Annual Rev. Biochem. 77:229-57. Lodish et al. Molecular Cell Biology. 1999. W. H. Freeman. 4th edition. Oldenburg et al. Recombination-mediated PCR directed plasmid construction in vivo in yeast. 1997. Nucleic Acids Research. Vol. 25, No. 2. Wyman et al. Homologous recombination-mediated double-strand break repair. (2004) DNA Repair. 3:827–833.

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