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Homologous Recombination

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

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Homologous Recombination

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

  3. Construction of P. falciparum expression vectors

  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 • No Holiday Junction formation • Break-induced repair • Telomeres • Replication forks

  5. Homologous Recombination Mechanism Filippo et al. 2008. 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

  8. http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=143http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=143

  9. http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=143http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=143

  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. 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.

  14. Questions?

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