Homologous Recombination: Presynaptic Filaments & Brca2 Scott Morrical Dept. of Biochemistry...
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Transcript of Homologous Recombination: Presynaptic Filaments & Brca2 Scott Morrical Dept. of Biochemistry...
Homologous Recombination:Presynaptic Filaments & Brca2
Scott MorricalDept. of [email protected]
1. E. coli RecA Paradigm2. Eukaryotic Rad513. Rad51-Brca2 Interactions
1: Bianco PR, Tracy RB, Kowalczykowski SC. DNA strand exchange proteins: a biochemical and physical comparison.Front Biosci. 1998 Jun 17;3:D570-603. Review. PMID: 9632377
2: Story RM, Weber IT, Steitz TA. The structure of the E. coli recA protein monomer and polymer.Nature. 1992 Jan 23;355(6358):318-25. Erratum in: Nature 1992 Feb6;355(6360):567. PMID: 1731246
3: Story RM, Steitz TA. Structure of the recA protein-ADP complex.Nature. 1992 Jan 23;355(6358):374-6. PMID: 1731253
4: Story RM, Bishop DK, Kleckner N, Steitz TA. Structural relationship of bacterial RecA proteins to recombination proteinsfrom bacteriophage T4 and yeast.Science. 1993 Mar 26;259(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 Aug;11(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 21;420(6913):287-93. Epub 2002 Nov 10. PMID: 12442171
Literature
Mitotic Recombination:Double-Strand Break Repair Model
ZAP!!
Broken Chromosome
Undamaged Homologous Chromosome
Nucleolytic Processing
DNA Strand Exchange (HR)
DNA Synthesis (RDR)
Repaired Chromosome
Endonucleolytic Resolution & Ligation
3’
3’
3’
3’
Types of DNA Rearrangements Catalyzed by E. coli RecA
2-strand reannealing:
+ATP ADP
3-strand exchanges:
ATP ADP+
ATP ADP
4-strand exchanges:
+ATP ADP
+
+ +
Properties of E. coli RecA Protein• Protomeric m.w. = 38 kDa.• Binds cooperatively to ssDNA at neutral pH; complex
stabilized by (d)ATP or ATPS, destabilized by ADP.• dsDNA binding requires low pH, ATPS, 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 theSOS response in E. coli.
Electron Micrograph of Relaxed Circular dsDNA Molecule Coatedwith RecA Protein in Presence of ATPS
• Open, right-handed helical filament• DNA is markedly extended and underwound
Story et al.: X-ray Crystallographic Structure of E. coli RecA-ADP Complex (Single Subunit Shown)
Presynaptic Filaments• RecA crystallizes as helical polymer even w/o DNA• DNA binding loops L1 & L2 are disordered
The RecA Paradigm of Homologous Strand Transfer
HomologousdsDNA
RecA
ATP, SSB
+
ssDNA
5’
3’
ATPADPATPADP
XRCC3
hRAD51B
hRAD51D
hRAD51C
UvsXRecA
hRAD51
hDMC1
RadA
XRCC2
Pf
Ec
Os
Ll2
Dr
Yp2
Uu
RadB
Pf
T4
Structure, Function & Evolution of DNA Repair Enzymes
Phylogenetic Diversity of RecA Family
RB69
RecA-dsDNA
(ATPS)
T4 UvsX-dsDNA
(ATP)
Yeast Rad51-dsDNA
(ADP-AlF4-)
Conservation of RecA Filament Structure
Modeling of Conserved Core Regions of T4 UvsX
and Yeast Rad51 onto Known X-ray Structure of
E. coli RecA
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 Aug;11(8):791-6.
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
Rad51 filament crystallized in presence of ssDNA oligo and ATPS
No ssDNA density.
Loops disordered.
SO42- occupies
nucleotide binding site.
Rad51 filament crystallized in presence of ssDNA oligo and ATPS
Exact 3-fold but only approximate 6-fold screw symmetry.
Alternating protomers are in different conformations! (Not seen in EcRecA structure)
H352A Mutation Destabilizes Yeast Rad51-ssDNA InteractionsConserved His in Rad51 and RadA branches;
Phe in bacterial RecAs
Tyr in T-even UvsXs
Rad51 filament crystallized in presence of ssDNA oligo and ATPS
Exact 3-fold but only approximate 6-fold screw symmetry.
Alternating protomers are in different conformations! (Not seen in EcRecA structure)
… But Evidence Suggests Brca2 Plays a Direct Role and Brca1 anIndirect Role in Promoting Rad51-Dependent Recombinational Repair
Brca1 Knockout Reduces Efficiency of Rad51 Repair FociFollowing Cisplatin or IR Exposure of Mouse ES Cells
Bishop & co-workers
IR-Induced Rad51 Foci Formation Requires Brca2(Spontaneous Rad51 Foci That Occur During S-Phase Are Brca2-Independent)
Cells contain Brca2mutant lacking nuclear localizationsignal; Brca2 stays in cytoplasm.
West
X-ray Structure of Human Rad51RecA Homology Domain
Complexed to Brca2 BRC Repeat
Pellegrini et al. (2002) Nature 420, 287-293
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!
BRC4
BRC4Rad51
Rad51
HsRad51vs.
EcRecA
The Rad51-BRC4 Interface
Hydrophobicinteractions via BRC4 -helix
Hydrophobicinteractions via BRC4 -hairpin
PolarInteractions
Brca2 Inhibits Rad51 Filament FormationCrystallographic EcRecA Filament
Superposition of BRC4 (from Rad51-BRC4 structure) on a subunit of EcRecA filament shows BRCA4 at interface between 2 EcRecA subunits.
EcRecA interface Rad51-BRC4 interface
EcRecA sequence26-IMRL-29 mediatespolymerization by anti-parallel -strand pairing
Brca2 sequence 1524-FHTA-1527 interactswith Rad51 by anti-parallel -strand pairing
Yeast Rad51 Interface (Conway et al.) Resembles Brca2 Peptide Interaction with Monomeric Human Rad51 (Pellegrini et al.)