Chapter 22 - Homologous recombination. The main contents of this chapter include all of the following: The RecBCD pathway for homologous recombination, experimental support for the RecBCD pathway, meiotic recombination, gene conversion. | Molecular Biology Fourth Edition Chapter 22 Homologous Recombination Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Homologous Recombination Pathways 22- RecBCD Pathway – Initial Binding RecBDC-sponsored homologous recombination in E. coli: DNA helicase activity unwinds the DNA toward a Chi-site Sequence 5’-GCTGGTGG-3’ Chi sites found on average every 5000 bp in E. coli genome RecBCD protein has ds- and ss-exonuclease activity ss-endonuclease activity Activities permit RecBCD to produce a ss-tail now coated by RecA protein 22- The RecBCD Pathway Schematic RecBCD pathway is a well-studied homologous recombination pathway used by E. coli 22- RecBCD Pathway – D Loop Invasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded break Invading strand forms a D loop (displacement) Loop is defined by displaced DNA strand When | Molecular Biology Fourth Edition Chapter 22 Homologous Recombination Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Homologous Recombination Pathways 22- RecBCD Pathway – Initial Binding RecBDC-sponsored homologous recombination in E. coli: DNA helicase activity unwinds the DNA toward a Chi-site Sequence 5’-GCTGGTGG-3’ Chi sites found on average every 5000 bp in E. coli genome RecBCD protein has ds- and ss-exonuclease activity ss-endonuclease activity Activities permit RecBCD to produce a ss-tail now coated by RecA protein 22- The RecBCD Pathway Schematic RecBCD pathway is a well-studied homologous recombination pathway used by E. coli 22- RecBCD Pathway – D Loop Invasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded break Invading strand forms a D loop (displacement) Loop is defined by displaced DNA strand When tail finds homologous region, nick occurs in in D-looped DNA Nick allows RecA and ss-break create a new tail that can pair with gap in the other DNA Subsequent degradation of the D-loop strand leads to the formation of a branched intermediate 22- Holliday Junctions Branch migration in this intermediate yields a Holliday junction with 2 strands exchanging between homologous chromosomes Branch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones in a process called branch migration This migration process does not occur at a useful rate spontaneously DNA unwinding required Unwinding requires helicase activity and energy from ATP 22- Resolving Holliday Junctions Holliday junctions can be resolved by nicking 2 of its strands Yielding: 2 noncrossover recombinant DNAs with patches of heteroduplex 2 crossover recombinant DNAs that have traded flanking DNA regions 22- Experimental Support for the RecBCD Pathway - RecA The .
