1. Microbial Structure and Function MI720 Fall 2007 DNA Recombination Andrew J. Pierce Department of Microbiology, Immunology and Molecular Genetics University of Kentucky

2. RecA Binds single-stranded DNA and double-stranded DNA Searches for regions of homology Exchanges homologous strands Image is from the cover of the March 26, 1993 issue of Science RecA + +

3. RecA homology search mechanism Flip the puckering of the ribose ring? PNAS Vol. 95, Issue 19, 11071-11076, September 15, 1998 Taro Nishinaka, Akira Shinohara, Yutaka Ito, Shigeyuki Yokoyama, and Takehiko Shibata

4. Substrates for Recombination Double-stranded DNA ends (double-strand breaks) Both strands broken Some intervening sequence missing Single-stranded DNA gaps One strand has sequence missing Problem: RecA can only load on single stranded DNA and has to compete with SSB Double-strand breaks use the RecBCD system Single-strand gaps use the RecFOR system

5. RecBCD Bind double-stranded DNA ends Degrade both stands until a  site (5' GCTGGTGG 3') is reached Switch to 5'-3' exonuclease generating a 3' single-stranded tail Load RecA on the single-stranded tail RecB:slow 3' to 5' helicase RecD:fast 5' to 3' helicase inhibits loading of RecA inactivated at  RecBC:loads RecA on constitutively Nature. 2004 Nov 11;432(7014):187-93. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Martin R. Singleton, Mark S. Dillingham, Martin Gaudier, Stephen C. Kowalczykowski and ﾊ Dale B. Wigley RecB RecC RecD

6. Model for RecBCD-mediated Repair  B D C 5' 3'  B D C 5' 3' A A A B D C 5' 3' A A A A invade homologous duplex 5' 3' A A A A A A A new DNA synthesis

7. Mol Cell. 2003 May;11(5):1337-47. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Morimatsu K, Kowalczykowski SC RecFOR RecF:bind single stranded gaps at the 5' boundary of the gap RecO:interact with and displace SSB load RecA RecR:bind to RecF recruit RecO (adaptor protein to limit the loading of RecO to single-strand gaps) RecA polymerizes 5' to 3'

8. Mol Cell. 2003 May;11(5):1337-47. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Morimatsu K, Kowalczykowski SC Model for RecFOR-mediated Repair

9. Mol Microbiol. 2004 Apr;52(2):5A-62. Requirement for RecFOR-mediated recombination in priA mutant. Grompone G, Sanchez N, Dusko Ehrlich S, Michel B. Bypassing a Lesion that Blocks Lagging Strand Synthesis

10. Bypassing a Lesion that Blocks Leading Strand Synthesis 3' 5' fork reversal reannealing duplex invasion A A A A A A A remove lesion B D C 3' 5' 3' 5' 3' 5' 3' 5' 3' 5' J Q

11. Recombination Between Bacterial Genomes: Bacterial Conjugation Involves the “F factor” aka “F plasmid” Non-Mendelian Inheritance F+ F-

12. Burland V, Shao Y, Perna NT, Plunkett G, Sofia HJ, Blattner FR. The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7. Nucleic Acids Res. 1998 Sep 15;26(18):4196-204. F Plasmids Can Carry Virulence Genes

13. F+ Can Recombine Into the Bacterial Chromosome At Regions of Homology Integration converts F+ to Hfr “Hfr” is High Frequency Recombination F+ Hfr

14. F' Hfr can excise from the chromosome taking some chromosomal DNA to form an F' episome F- Hfr Hfr Can Transfer Entire Bacterial Genome to Recipient Cell or Excise as an F' Episome transfer is very rarely complete, but all transferred sequence can recombine F- still F- ( since F+ genes transfer last )