Microbial Structure and Function

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Microbial Structure and Function
MI720
Fall 2007
DNA Recombination
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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
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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
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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

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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 11432(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?Dale B. Wigley
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Model for RecBCD-mediated Repair
C
5'
3'
C
B
C
D
B
C
D
invade homologous duplex
new DNA synthesis
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RecFOR
RecA polymerizes 5' to 3'
Mol Cell. 2003 May11(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
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Model for RecFOR-mediated Repair
Mol Cell. 2003 May11(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
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Bypassing a Lesion that Blocks Lagging Strand
Synthesis
Mol Microbiol. 2004 Apr52(2)5A-62. Requirement
for RecFOR-mediated recombination in priA
mutant. Grompone G, Sanchez N, Dusko Ehrlich S,
Michel B.
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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'
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Recombination Between Bacterial
Genomes Bacterial Conjugation

• Involves the F factor aka F plasmid
• Non-Mendelian Inheritance

F
F-
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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 O157H7. Nucleic Acids Res. 1998
Sep 1526(18)4196-204.
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F Can Recombine Into the Bacterial Chromosome At
Regions of Homology

• Integration converts F to Hfr
• Hfr is High Frequency Recombination

Hfr
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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