Homologous Recombination:Presynaptic Filaments & Brca2

Scott MorricalDept. of Biochemistrysmorrica@zoo.uvm.edu

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

Holliday Modelof HomologousGeneticRecombination

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’

Recombination Lessons fromProkaryotes:

The E. coli RecA Paradigm

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

RecA Filament Structure

Key Residues of E. coli RecA

The RecA Paradigm of Homologous Strand Transfer

HomologousdsDNA

RecA

ATP, SSB

+

ssDNA

5’

3’

ATPADPATPADP

Other Factors Involved in Homologous Recombination in E. coli

(Mediators)

Conservationof RecA Family

in Diverse Species

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

Sequence Alignment of E. coli RecA, Yeast Rad51, & T4 UvsX

Modeling of Conserved Core Regions of T4 UvsX

and Yeast Rad51 onto Known X-ray Structure of

E. coli RecA

UvsXvs.

RecA

Rad51vs.

RecA

Identical Residues Mapped on E. coli RecA Structure

Conservation of Other Recombination Functionalities

(Mediators)

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)

Two Types of Protomer-ProtomerInteractions at Rad51 ATPase Site

H352A Mutation Destabilizes Yeast Rad51-ssDNA InteractionsConserved His in Rad51 and RadA branches;

Phe in bacterial RecAs

Met in T-even UvsXs

Tyrosine Phosphorylation Site at Protomer-

Protomer Interface

Relationship ofBRCA Gene Products

to Homologous Recombination& DSBR

Nobody Said It Would Be Simple…

… 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.)

Mutant GFP-Fusion Rad51 Proteins F86E & A89E Designed toDisrupt Hydrophobic Contacts in Putative Rad51 Subunit Interface

Result: WT but not mutant Rad51s can pull down endogenous WTRad51 in co-IP expts. Ability to interact with Brca2 remains intact.

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

Brca2: Designed to Load Rad51 Onto ssDNA?

Multiple BRC RepeatsIn Brca2 Could Serveas a Pre-Loading &Assembly Site for Rad51,All Ready for Transfer Onto ssDNA Bound toOB-folds in the DNABinding Domain

3HB Motif in TowerDomain: Tether Complexto Duplex Portion ofTailed DSB???

Why Are Defects MainlyAssociated With Tumorsof Breast & Ovary???