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Microbial Structure and Function

Andrew J. Pierce. Department of Microbiology, Immunology and Molecular Genetics. University of Kentucky. Microbial Structure and Function. MI720. Fall 2007. DNA Recombination. RecA. Binds single-stranded DNA and double-stranded DNA Searches for regions of homology

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Microbial Structure and Function

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

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

  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. RecB: slow 3' to 5' helicase RecD: fast 5' to 3' helicase inhibits loading of RecA inactivated at C RecBC: loads RecA on constitutively RecB RecC RecD RecBCD • Bind double-stranded DNA ends • Degrade both stands until a C site (5' GCTGGTGG 3') is reached • Switch to 5'-3' exonuclease generating a 3' single-stranded tail • Load RecA on the single-stranded tail 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

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

  7. RecF: bind single stranded gaps at the 5' boundary of the gap RecR: bind to RecF recruit RecO (adaptor protein to limit the loading of RecO to single-strand gaps) RecO: interact with and displace SSB load RecA RecFOR RecA polymerizes 5' to 3' 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

  8. Model for RecFOR-mediated Repair 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

  9. Bypassing a Lesion that Blocks Lagging Strand Synthesis 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.

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

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

  12. F Plasmids Can Carry Virulence Genes 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.

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

  14. F' 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 Hfr Hfr F- F- still F- ( since F+ genes transfer last ) • Hfr can excise from the chromosome taking some chromosomal DNA to form an F' episome Hfr

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