Download Bacterial Recombination

Microbial Structure and Function
MI720
Fall 2007
DNA Recombination
Andrew J. Pierce
Department of Microbiology, Immunology and Molecular Genetics
University of Kentucky
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
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
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
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
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
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
Model for RecBCD-mediated Repair
B
C
D
C
5'
3'
C
5'
3'
5'
3'
B
C
D
B A AA A A A A
C
D
invade homologous duplex
5'
3'
new DNA synthesis
A AA
AA
A
A
RecFOR
RecA polymerizes
5' to 3'
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
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
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
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.
Bypassing a Lesion that Blocks
Leading Strand Synthesis
3'
5'
3'
5'
remove lesion
3'
5'
J
Q
B
D
C
fork
reversal
reannealing
3'
5'
3'
5'
duplex invasion
A AA
3'
5'
AA
A
A
Recombination Between Bacterial Genomes:
Bacterial Conjugation
• Involves the “F factor” aka “F plasmid”
• Non-Mendelian Inheritance
F+
F-
F+
F+
F+
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.
F+ Can Recombine Into the Bacterial Chromosome
At Regions of Homology
• Integration converts F+ to Hfr
• “Hfr” is High Frequency Recombination
Hfr
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-
• Hfr can excise from the chromosome taking some
chromosomal DNA to form an F' episome
Hfr
F'
still F( since F+ genes
transfer last )