DNA%20Recombination

1
DNA Recombination

• Roles
• Types
• Homologous recombination in E.coli
• Transposable elements

2
Biological Roles for Recombination

1. Generating new gene/allele combinations
   (crossing over during meiosis)
2. Generating new genes (e.g., Immuno- globulin
   rearrangement)
3. Integration of a specific DNA element (or
   virus)
4. DNA repair

3
Practical Uses of Recombination

• Used to map genes on chromosomes
• - recombination frequency proportional to
  distance between genes
• 2. Making transgenic cells and organisms

4
Map of Chromosome I of Chlamydomonas reinhardtii
cM centiMorgan unit of recombination
frequency 1 cM 1 recombination frequency
Chlamydomonas Genetics Center
5
Types of Recombination

1. Homologous - occurs between sequences that are
   nearly identical (e.g., during meiosis)
2. Site-Specific - occurs between sequences with a
   limited stretch of similarity involves specific
   sites
3. Transposition DNA element moves from one site
   to another, usually little sequence similarity
   involved

6
Examples of (mostly) Homologous Recombination
Fig. 22.1
7

• Holliday Model
• R. Holliday (1964)
• Holliday Junctions form during recombination
• HJs can be resolved 2 ways, only one produces
  true recombinant molecules

patch
8
EM of a Holliday Junction w/a few melted base
pairs around junction
Fig. 22.3
9
The recBCD Pathway of Homologous Recombination
Part I Nicking and Exchanging
Fig. 22.2 a-d
10
recBCD Pathway of Homologous Recomb. Part I
Nicking and Exchanging

1. A nick is created in one strand by recBCD at a
   Chi sequence (GCTGGTGG), found every 5000 bp.
2. Unwinding of DNA containing Chi sequence by
   recBCD allows binding of SSB and recA.
3. recA promotes strand invasion into homologous
   DNA, displacing one strand.
4. The displaced strand base-pairs with the single
   strand left behind on the other chromosome.
5. The displaced and now paired strand is nicked
   (by recBCD?) to complete strand exchange.

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recBCD Pathway of Homologous Recombination Part
II Branch Migration and Resolution
Fig. 22.5 f-h
12
recBCD Pathway of Homologous Recom. Part II
Branch Migration and Resolution

• Nicks are sealed ? Holliday Junction
• Branch migration (ruvA ruvB)
• Resolution of Holliday Junction (ruvC)

13
RecBCD A Complex Enzyme

• RecBCD has
• Endonuclease subunits (recBC) that cut one DNA
  strand close to Chi sequence.
• DNA helicase activity (recD subunit) and
• a DNA-dependent ATPase activity
• unwinds DNA to generate the 3 SS tails

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RecA

• 38 kDa protein that polymerizes onto SS DNA 5-3
• Catalyzes strand exchange, also an ATPase
• Also binds DS DNA, but not as strongly as SS

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RecA binds preferentially to SS DNA and will
catalyze invasion of a DS DNA molecule by a SS
homologue. Important for many types of
homologous recombination, such as during meoisis
(in yeast).
Fig. 6.19 in Buchanan et al.
16
RecA Function Dissected

• 3 steps of strand exchange
• Pre-synapsis recA coats single-stranded DNA
  (accelerated by SSB, so get more relaxed
  structure).
• Synapsis alignment of complementary sequences in
  SS and DS DNA (paranemic or side-by-side
  structure).
• Post-synapsis or strand-exchange SS DNA replaces
  the same strand in the duplex to form a new DS
  DNA (requires ATP hydrolysis).

17
RuvA and RuvB

• DNA helicase that catalyzes branch migration
• RuvA tetramer binds to HJ (each DNA helix
  between subunits), forces it into square planar
  conformation
• 2 copies of RuvB bind at the HJ (to RuvA and 2
  of the DNA helices)
• RuvB is a hexamer ring, has helicase ATPase
  activity
• Branch migration is in the direction of recA
  mediated strand-exchange

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RuvA/RuvB/DNA Complex
RuvB
RuvA
Shows RuvB encircling DNA duplexes
19
RuvB
RuvA
RuvA removed for visual purposes only
Similar to Figure 22.13 Model based on EM
images.
20
RuvC resolvase

• Endonuclease that cuts 2 strands of HJ
• Binds to HJ as a dimer (that already has
  RuvA/RuvB)
• Consensus sequence (A/T)TT (G/C)
• - occurs frequently in E. coli genome
• - branch migration needed to reach consensus
  sequence!

21
RuvC bound to a HJ
Fig. 22.16
22
A model for binding of RuvA, RuvB, and RuvC to a
HJ.
Fig. 22.17b
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Meiotic Recomb. in Yeast- is initiated by a
double-strand break (DSB)
Fig. 22.18
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Repair of double-strand breaks (DSBs)in
non-dividing or mitotic cells

• DSBs probably most severe form of DNA damage, can
  cause loss of genes or even cell death
  (apoptosis)
• DSBs caused by
• - ionizing radiation
• - certain chemicals
• - some enzymes (topoisomerases, endonucleases)
• - torsional stress

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2 general ways to repair DSBs

• Homologous recombination (HR) - repair of broken
  DNA using the intact homologue, very similar to
  meiotic recombination. Very accurate.
• Non-homologous end joining (NHEJ) - ligating
  non-homologous ends. Prone to errors, ends can be
  damaged before religation (genetic material lost)
  or get translocations. (Mechanism in Fig 20.38)
• Usage NHEJ gtgt HR in plants and animals