Homologous Recombination:

1

• Homologous Recombination
• Presynaptic Filaments Brca2
• Scott Morrical
• Dept. of Biochemistry
• smorrica_at_zoo.uvm.edu
• E. coli RecA Paradigm
• Eukaryotic Rad51
• Rad51-Brca2 Interactions

2
Literature
1 Bianco PR, Tracy RB, Kowalczykowski SC. DNA
strand exchange proteins a biochemical and
physical comparison. Front Biosci. 1998 Jun
173D570-603. Review. PMID 9632377 2 Story
RM, Weber IT, Steitz TA. The structure of the
E. coli recA protein monomer and polymer. Nature.
1992 Jan 23355(6358)318-25. Erratum in Nature
1992 Feb 6355(6360)567. PMID 1731246 3
Story RM, Steitz TA. Structure of the recA
protein-ADP complex. Nature. 1992 Jan
23355(6358)374-6. PMID 1731253 4 Story RM,
Bishop DK, Kleckner N, Steitz TA. Structural
relationship of bacterial RecA proteins to
recombination proteins from bacteriophage T4 and
yeast. Science. 1993 Mar 26259(5103)1892-6.
PMID 8456313 5 Conway AB, Lynch TW, Zhang Y,
Fortin GS, Fung CW, Symington LS, Rice PA.
Crystal structure of a Rad51 filament. Nat Struct
Mol Biol. 2004 Aug11(8)791-6. Epub 2004 Jul 4.
PMID 15235592 6 Pellegrini L, Yu DS, Lo T,
Anand S, Lee M, Blundell TL, Venkitaraman AR.
Insights into DNA recombination from the
structure of a RAD51-BRCA2 complex. Nature. 2002
Nov 21420(6913)287-93. Epub 2002 Nov 10. PMID
12442171
3
Holliday Model of Homologous Genetic Recombination
4
Mitotic Recombination Double-Strand Break Repair
Model
ZAP!!
Broken Chromosome
Nucleolytic Processing
3
3
DNA Strand Exchange (HR)
3
3
Undamaged Homologous Chromosome
DNA Synthesis (RDR)
Endonucleolytic Resolution Ligation
Repaired Chromosome
5
Recombination Lessons from Prokaryotes The E.
coli RecA Paradigm
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Types of DNA Rearrangements Catalyzed by E. coli
RecA
2-strand reannealing

3-strand exchanges



4-strand exchanges


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Properties of E. coli RecA Protein

• Protomeric m.w. 38 kDa.
• Binds cooperatively to ssDNA at neutral pH
  complex
• stabilized by (d)ATP or ATPgS, destabilized by
  ADP.
• dsDNA binding requires low pH, ATPgS, or
  transfer or
• nucleation from ssDNA.
• Forms filaments on off of DNA.
• Presynaptic filament-- RecA filament assembled
  on ssDNA in
• presence of Mg(d)ATP-- is catalytically active
  form.
• Catalyzes DNA-dependent (d)ATP hydrolysis.
• Catalyzes (d)ATP-dependent DNA rearrangements
  including
• complementary strand reannealing homologous 3-
  or
• 4-strand strand exchanges.
• Co-protease In response to DNA damage,
  facilitates auto-
• proteolytic cleavage of LexA repressor which
  induces the
• SOS response in E. coli.

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Electron Micrograph of Relaxed Circular dsDNA
Molecule Coated with RecA Protein in Presence of
ATPgS

• Open, right-handed helical filament
• DNA is markedly extended and underwound

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Story et al. X-ray Crystallographic Structure
of E. coli RecA-ADP Complex (Single Subunit
Shown)

• RecA crystallizes as helical polymer even w/o
  DNA
• DNA binding loops L1 L2 are disordered

Presynaptic Filaments
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RecA Filament Structure
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Key Residues of E. coli RecA
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The RecA Paradigm of Homologous Strand Transfer
Presynapsis
RecA
Homologous dsDNA
ssDNA
ATP, SSB
3
Synapsis
5

ATP
ADP
ATP
ADP
Branch Migration
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Other Factors Involved in Homologous
Recombination in E. coli
(Mediators)
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Conservation of RecA Family in Diverse Species
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Phylogenetic Diversity of RecA Family
RadA
Pf
hDMC1
Yp2
hRAD51
XRCC3
XRCC2
Uu
hRAD51B
Ll2
RB69
hRAD51D
Pf
Dr
hRAD51C
RadB
T4
Ec
UvsX
RecA
Os
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Conservation of RecA Filament Structure
RecA-dsDNA (ATPgS)
T4 UvsX-dsDNA (ATP)
Yeast Rad51-dsDNA (ADP-AlF4-)
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Sequence Alignment of E. coli RecA, Yeast Rad51,
T4 UvsX
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Modeling of Conserved Core Regions of T4 UvsX and
Yeast Rad51 onto Known X-ray Structure of E. coli
RecA
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Identical Residues Mapped on E. coli RecA
Structure
UvsX vs. RecA
Rad51 vs. RecA
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Conservation of Other Recombination
Functionalities
(Mediators)
21
Crystal structure of a Rad51 filament. Conway
AB, Lynch TW, Zhang Y, Fortin GS, Fung CW,
Symington LS, Rice PA. Nat Struct Mol Biol. 2004
Aug11(8)791-6.
22
N-terminally Truncated Yeast Rad51 (the form used
for crystallization) Catalyzes Strand Exchange
Crystallized form also contained I345T mutation--
a gain-of-function mutation, enhanced
ssDNA-binding form, suppresses rad55/57 mediator
mutations
23
Rad51 filament crystallized in presence of ssDNA
oligo and ATPgS
No ssDNA density. Loops disordered. SO42-
occupies nucleotide binding site.
24
Rad51 filament crystallized in presence of ssDNA
oligo and ATPgS
Exact 3-fold but only approximate 6-fold screw
symmetry. Alternating protomers are in different
conformations! (Not seen in EcRecA structure)
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Two Types of Protomer-Protomer Interactions at
Rad51 ATPase Site
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H352A Mutation Destabilizes Yeast Rad51-ssDNA
Interactions Conserved His in Rad51 and RadA
branches Phe in bacterial RecAs Met in T-even
UvsXs
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Tyrosine Phosphorylation Site at
Protomer-Protomer Interface
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Relationship of BRCA Gene Products to Homologous
Recombination DSBR
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Nobody Said It Would Be Simple
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But Evidence Suggests Brca2 Plays a Direct Role
and Brca1 an Indirect Role in Promoting
Rad51-Dependent Recombinational Repair
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Brca1 Knockout Reduces Efficiency of Rad51 Repair
Foci Following Cisplatin or IR Exposure of Mouse
ES Cells
Bishop co-workers
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IR-Induced Rad51 Foci Formation Requires
Brca2 (Spontaneous Rad51 Foci That Occur During
S-Phase Are Brca2-Independent)
Cells contain Brca2 mutant lacking nuclear
localization signal Brca2 stays in
cytoplasm. West
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X-ray Structure of Human Rad51 RecA Homology
Domain Complexed to Brca2 BRC Repeat Pellegrini
et al. (2002) Nature 420, 287-293
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1.7 Å Structure of Human BRCA Repeat 4 (A.A.
1517-1551) Bound to RecA Homology Domain of Rad51
(S95 - C-Terminus) An Ingenious Trick BRC4
fused to N-terminus of truncated Rad51 via
flexible linker-- suppresses natural tendency of
Rad51 to self-aggregate!
Rad51
Rad51
BRC4
BRC4
HsRad51 vs. EcRecA
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The Rad51-BRC4 Interface
Hydrophobic interactions via BRC4 a-helix
Hydrophobic interactions via BRC4 b-hairpin
Polar Interactions
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Brca2 Inhibits Rad51 Filament Formation
Crystallographic EcRecA Filament
Superposition of BRC4 (from Rad51-BRC4 structure)
on a subunit of EcRecA filament shows BRCA4 at
interface between 2 EcRecA subunits.
EcRecA sequence 26-IMRL-29 mediates polymerization
by anti- parallel b-strand pairing Brca2
sequence 1524-FHTA-1527 interacts with Rad51 by
anti- parallel b-strand pairing
EcRecA interface
Rad51-BRC4 interface
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Yeast Rad51 Interface (Conway et al.) Resembles
Brca2 Peptide Interaction with Monomeric Human
Rad51 (Pellegrini et al.)
38
Mutant GFP-Fusion Rad51 Proteins F86E A89E
Designed to Disrupt Hydrophobic Contacts in
Putative Rad51 Subunit Interface
Result WT but not mutant Rad51s can pull down
endogenous WT Rad51 in co-IP expts. Ability to
interact with Brca2 remains intact.
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• Impacts of Rad51 Polymerization Mutants F86E
  A89E on Ability
• to Form Nuclear Foci in Dividing 293T Cells or
  Following IR Exposure
• Mutants inhibit foci formation
• So does overexpression of BRC3/4 repeat fragment

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Brca2 Designed to Load Rad51 Onto ssDNA?
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Multiple BRC Repeats In Brca2 Could Serve as a
Pre-Loading Assembly Site for Rad51, All Ready
for Transfer Onto ssDNA Bound to OB-folds in the
DNA Binding Domain 3HB Motif in Tower Domain
Tether Complex to Duplex Portion of Tailed
DSB??? Why Are Defects Mainly Associated With
Tumors of Breast Ovary???