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Homologous Recombination &
Double-Strand Break Repair:
Crossing-Over with Cancer Biology
Scott Morrical
Dept. of Biochemistry
[email protected]
1. Lessons from Prokaryotes & Yeast
2. DSBR in Humans-- Mediators, Paralogs, & BRCA1/2
Types of Recombination:
1. Site-specific recombination.
Recombination occurs at defined, short sequences in DNA.
Requires a site-specific recombinase enzyme that recognizes
the target sequence.
2. Non-homologous or illegitimate recombination.
Little or no sequence specificity or homology requirement.
Certain types of transposition; non-homologous end joining.
3. General or homologous recombination.
Occurs between any homologous DNA sequences of sufficient
length. Meiotic crossing-over; DNA repair.
Homologous Recombination & Cancer:
Why You Should Care
1. Homologous recombination is required for accurate repair
of DNA double-strand breaks (DSBs). Therefore protective
against carcinogenesis. Errors --> increased mutation rates
& susceptibility to carcinogenesis.
Nijmegen chromosome breakage syndrome (NBS)
Ataxia telangiectasia (AT)
AT-like disorder (ATLD)
Bloom’s syndrome (BLM)
Fanconi's anemia (FA)
Werner’s syndrome (WRN)
2. Functions of human breast/ovarian cancer susceptibility
genes BRCA1 & BRCA2 are clearly linked to homologous
recombination and double-strand break repair.
3. Aberrant recombination phenotypes associated with
neoplastic states-- i.e. hyper-recombination in p53 mutants.
4. Homologous recombination & DSBR are mechanisms of
tumor cell resistance to radiation and chemotherapy. Targeting
recombination pathways in tumor cells could increase efficacy.
5. Targeted homologous recombination is desirable for cancer
gene therapy approaches, i.e. introduction of suicide genes at
benign locations in the genome.
Holliday Model
of Homologous
Genetic
Recombination
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
Recombination Lessons from
Prokaryotes:
The E. coli RecA Paradigm
Phylogenetic Diversity of RecA Family
RadA
hDMC1
Yp2
Pf
hRAD51
XRCC3
XRCC2
Uu
hRAD51B
Ll2
RB69
T4
Pf
Dr
RadB
hRAD51D
hRAD51C
Ec
UvsX
Os
RecA
Structure, Function
& Evolution of
DNA Repair Enzymes
Types of DNA Rearrangements Catalyzed by E. coli RecA
2-strand reannealing:
ATP
ADP
+
3-strand exchanges:
ATP
+
ADP
ATP
+
+
4-strand exchanges:
+
ATP
ADP
+
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 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 autoproteolytic cleavage of LexA repressor which induces the
SOS response in E. coli.
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
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
The RecA Paradigm of Homologous Strand Transfer
RecA
ssDNA
Homologous
dsDNA
ATP, SSB
3’
5’
+
ADP
ATP
ADP
ATP
Other Recombination Proteins Affect
DNA Strand Exchange
• Nucleases/helicases generate ssDNA substrates for
presynapsis.
• ssDNA-binding proteins (SSBs)-- promote presynapsis*,
sequester displaced strand in branch migration.
• Recombination mediator proteins (RMPs)-- assemble
presynaptic filament.
• DNA helicases/translocases-- promote branch migration,
filament remodeling.
Problems in Presynaptic Filament Assembly:
• Targeting filament assembly onto ssDNA in the presence of
excess cellular dsDNA.
• Competition between RecAs and abundant cellular SSBs for
binding to ssDNA.
Order of Addition Effect:
-- SSB added to ssDNA after preincubation of RecA + ssDNA + ATP
gives optimal stimulation of filament assembly, ATPase, & strand
exchange activities.
-- SSB preincubated with ssDNA before RecA + ATP added gives
strong inhibition of filament assembly, ATPase, & strand exchange.
Both problems dealt with by Recombination
Mediator Proteins (RMPs) & other factors
Evolutionary Conservation of Recombinase, SSB,
& Mediator Functionalities
Gp32
T4 UvsX-ssDNA
Presynaptic
Filaments
UvsX
T4 phage
E. coli
S. cerevisiae
H. sapiens
Recombinase:
UvsX
RecA
Rad51
Rad51
SSB:
Gp32
SSB
RP-A
RP-A
Mediator(s):
UvsY
RecO/R
RecF?
Rad52
Rad55/57
Rad52
Rad51B,C,D?
Xrcc2,3?
Brca2?
Enzymology of DSBR
• Yeast RAD52 Epistasis Group
• Human Rad51 paralogs
• The BRCA connection
How (Unprogrammed) DNA Double-Strand Breaks Occur
1. Ionizing Radiation (i.e. X- & g-rays) and some chemical
agents locally disrupt the backbones of both strands of B-form
DNA.
2. Inappropriate cleavage of dsDNA by an endonuclease.
3. BER or NER enzyme processing of interstrand crosslinks or
of base lesions too close to nicks on the opposite strand.
4. Replication fork collapse:
--Replication past a single-strand disruption or nick.
--Replication fork collisions with cleavage complexes of
type I & II topoisomerases.
5. Deoxyribonucleotide starvation.
Implications for Cancer Treatment
• Radiation: Hope that rapidly proliferating tumor cells won’t be
able to repair induced DSBs fast enough, & therefore selectively
undergo apoptosis. Problems-- resistant cells are good at DSBR;
doesn’t work well for slower-growing tumors; secondary effects.
• Topoisomerase poisons: Stabilize topo-DNA cleavage complexes,
increase frequency of replication fork collapse in rapidly proliferating
tumor cells.
nick
+
Topo-II
+ m-AMSA
Topo-I
+ camptothecin
+
?
+
• Hydroxyurea: Inhibitor of ribonucleotide reductase.
Chemotherapy deprives rapidly proliferating tumor cells of
deoxyribonucleotide precursors for DNA synthesis & repair.
Observation: DSBs accumulate in treated cells- why?
Many stalled replication forks; get converted into
mitotic DSBs (?)
Inability to complete replicative steps of DSBR
pathways.
RAD52 Epistasis Group
In Yeast (& Humans)
Genetically Implicated
in Homologous Recombination
& Double-Strand Break Repair
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
RAD52 Epistasis Group: Genes & Gene Products
(All conserved in humans in one way or another)
Processing:
MRE11
Mre11/Rad50/Xrs2 complex (MRX)
RAD50
implicated in nucleolytic resection of
XRS2 (NBS1)
DSBs --> 3’ ssDNA tails
Recombination:
RAD51
Ortholog of E. coli RecA
RAD52
Mediator, annealing & strand exchange protein
RAD54
Snf2/Swi2 ATPase
RAD55
Rad51 paralogs; Rad55/Rad57 dimer = mediator
RAD57
(Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3)
RAD59
Rad52 paralog
RDH54
Rad54 paralog
RFA1
Lg. Subunit of RPA (SSB) heterotrimer
Mediator
Rings &
Oligomers
“7-11”
Rad52
Single-strand
Annealing
Strand
Exchange
Mediator Function
Of Yeast Rad52
Yeast Rad52 relieves RPA order of
addition effect in Rad51-catalyzed
DNA strand exchange assay…
Rad51 -> RPA
RPA+Rad52 -> Rad51
RPA -> Rad51
72 min
…but Rad52 does not replace RPA
in strand exchange; rxns remain
RPA-dependent.
36 min
Biochemical Demonstration of Yeast
Rad51-Rad52 Interactions
Immunoprecipitations:
From wt extracts
Affinity Chromatography:
From Rad52 overexpresser
Sung et al.
N-terminal Fragment of Human Rad52 (Residues 1-209) Promotes
Reannealing & Crystallizes as an Undecameric Ring
bbba
I
Singleton et al.; Kagawa et al.
II
Propagation of Putative ssDNA Binding Site
Around the Ring Surface of HsRad521-209
Yeast Rad54
• Member of Snf2/Swi2 family of DNA-dependent
ATPases/motor proteins/helicases.
• Binds to dsDNA and introduces local and global changes
in superhelicity consistent with translocation along duplex
without unwinding.
• Binds to Rad51 and stimulates DNA strand exchange rxns.
• Overcomes dsDNA inhibition of Rad51-catalyzed DNA
strand exchange.
Rad51 differs from E. coli RecA in having an
intrinsically high affinity for dsDNA-- the dsDNA
can actually sequester Rad51 & thereby inhibit
strand exchange initiated from ssDNA.
Heyer & co-workers:
Rad54 Disassembles
Inappropriate Rad51dsDNA Complexes
& Thereby Facilitates
Appropriate ssDNAInitiated DNA Strand
Exchanges
Rad54 may also
promote nucleosome
rearrangements
around target sequence
in homologous
duplex.
Mre11/Rad50/Xrs2 (MRX) Complex
(Yeast & Human Versions)
• Localizes with nuclear “repair foci” following cell
exposure to ionizing radiation.
• Implicated in resection of DSBs into 3’ ssDNA tails
-- curious, since Mre11 is a weak 3’ --> 5’ exonuclease!
-- Mre11 also has ssDNA endonuclease activity
-- all Mre11 nuclease activities Mn++ dependent
• Mre11 & Rad50 are conserved in all kingdoms of life.
Xrs2 is only weakly conserved. Human Mre11/Rad50
complex associates with Nbs1, which is deficient in NBS, a
rare cancer-prone syndrome. Hypomorphic alleles of Mre11
cause A-TLD, a human chromosomal instability syndrome.
Proposed Role of MRX in DSB Resection in Yeast
Wild-type MRX: weak unwinding activity or recruits helicase.
mre11-H125N: still unwinds but lacks ssDNA endonuclease.
Electron
Microscopy
Of Yeast
Rad50-Mre11
Complexes
Rad50
+
Mre11
(2:2)
(2:1)
Mre11
Anderson et al., J. Biol. Chem., Vol. 276, Issue 40, 37027-37033, October 5, 2001
Rad50: Member of SMC Family. Walker A & B ATP-Binding
Motifs Separated by Long Coiled-Coil Domain, Used (?) to Orient
Mre11 Subunits & Link DSB Sites
Human (Vertebrate)
Rad51 Paralogs:
Rad51B
Rad51C
Rad51D
Xrcc2
Xrcc3
Implicated in Homologous Recombination
& Repair; Formation of Nuclear Rad51 Foci
Following IR Exposure, Etc.
Rad51B Knockouts in Chicken B Lymphocyte DT40 Cells
Compromise Rad51 Repair Foci Induced by DNA Damaging Agents
Takata et al.
Human Rad51 Paralogs Form
Two Distinct Complexes:
(West, Sung, & other labs)
BCDX2
CX3
BCDX2-- 1:1:1:1 Stoichiometry
CX3-- 1:1 Stoichiometry
Summary:
Biochemical Activities Ascribed to Rad51 Paralog Assemblies
& Sub-Assemblies
Complex
ss-Binding
ATPase
B
C
D
X2
BC
DX2
BCDX2
X3
CX3
X (+ HJ, duplex)
X
X
X
X
X
X
X (+ duplex)
X (+ nicks)
X
X
Mediator*
Strand Ex*
X
X (no ATP?)
X (Rad51 ATP/ADP exchange)
X
X (no ATP?)
X
X (no ATP?)
*Caution necessary since C and CX3 have weak strand exchange activities.
Role of Breast/Ovarian Cancer
Susceptibility Genes
BRCA1 & BRCA2
In Homologous Recombinatin
& DNA Repair
Nobody Said It Would Be Simple…
… But Evidence Suggests Brca2 Plays a Direct Role and Brca1 an
Indirect Role in Promoting Rad51-Dependent Recombinational Repair
Brca1 Knockout Reduces Efficiency of Rad51 Repair Foci
Following 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 Brca2
mutant lacking
nuclear localization
signal; Brca2 stays
in cytoplasm.
West
X-ray Structure of Mouse/Rat
Brca2 ssDNA-Binding Domain
Complexed to Dss1 & ssDNA
Yang et al. (2002) Science 297, 1837-1848
Structure of
Mouse Brca2
DNA-Binding
Domain:
D = Intact DBDDss1 complex
E = Tower deletion DBD
mutant bound to Dss1 & oligo dT9
OB-fold: Oligonucleotide/
oligosaccharide binding fold,
structurally conserved.
Brca2DBDDTower-Dss1-dT9 Complex at 3.5 Å
5 of 9 ssDNA Residues Resolve, Bound Across OB2 & OB3
X-ray Structure of Human Rad51
RecA 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!
Rad51
Rad51
BRC4
BRC4
HsRad51
vs.
EcRecA
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 antiparallel b-strand pairing
Brca2 sequence
1524-FHTA-1527 interacts
with Rad51 by antiparallel b-strand pairing
EcRecA interface
Rad51-BRC4 interface
Brca2: Designed to Load Rad51 Onto ssDNA?
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???
Other Cancer-Predisposition Syndromes:
1. Ataxia telangiectasia (AT)
Symptoms:
-- progressive neuronal degeneration, loss of cerebellar function
-- immunodeficiency, sterility, clinical radiation sensitivity
-- 60-180x increase in malignancies (70% lymphomas and T-cell leuk.)
Cellular
Phenotype
-- chromosomal breakage, telomere instability, radiosensitivity
Defective
Gene
-- ATM (ataxia telangiectasia mutated)
-- radioresistant DNA synthesis, defective cell cycle checkpoints
-- dysfunctional apoptosis, reduced p53 response
-- residual unrepaired DSBs
-- 3056 a.a. ATM protein is member of phosphoinositol 3-kinase family
-- master regulator in a signaling network responsible for coordinating
DSB repair, checkpoint functions, & other signaling processes that
promote cellular recovery and survival
DSB-induced
phosphorylation rxns
mediated by the ATM
kinase that lead to
transcriptional changes,
implementation of cell
cycle checkpoints, and
execution of DNA repair
processes.
Other Cancer-Predisposition Syndromes:
2. Nijmegen breakage syndrome (NBS)
Symptoms:
-- resembles AT but lacks ataxia and telangiectasia
-- immunodeficiency, radiation sensitivity
-- predisposition to malignancies
Cellular
Phenotype
-- similar to AT
Defective
Gene
-- NBS1
-- 754 a.a. NBS1 protein is a component of Mre11-Rad50-Nbs1
complex that is implicated in processing DSBS into 3’ ssDNA tails.
-- Phosphorylation at Ser343 and other sites by ATM kinase is
necessary for IR resistance.
Domain Structure of Nbs1
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N-terminus
FHA (forkhead-associated) / BRCT (Brca1 c-terminal) domain
mediates association of Nbs1 with histone g-H2AX, which is
subsequently phosphorylated by ATM kinase.
Function of NBS1 in
rejoining of DSBs and cell
cycle checkpoint control.
NBS1 is recruited to
damaged sites by binding
to MRE11 in an ATMindependent manner and
subsequently, the MRN
complex recruits ATM
kinase to such sites and
H2AX is phosphorylated
by ATM and other
members of the ATMrelated protein kinases.
The phosphorylation of
H2AX recruits/retains more
MRN complex at damaged
sites, and initiates HR
repair.
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Other Cancer-Predisposition Syndromes:
3. Fanconi’s anemia (FA)
Symptoms:
-- predisposition to malignancies, espeically acute myeloid leukemia
(15,000x increase), squamous cell carcinoma (avg. onset age = 24 yrs)
-- progressive aplastic anemia caused by loss of bone marrow stem cells
-- diverse developmental abnormalities
Cellular
Phenotype
-- chromosomal sensitivity to crosslinking reagents
Defective
Genes
-- FancA, FancC, FancE, FancF, FancG --> nuclear complex
-- FancD2 --> ubiquitination target; phosphorylated by ATM kinase
-- FancB = FancD1 = BRCA2!!!!!
Interactions between the FA proteins and their potential roles in DNA repair.
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BRCA1 is required for ubiquitination of FANCD2. The activated FANCD2 protein is then seen
to colocalize with BRCA1 in nuclear foci, where it may interact with other repair proteins.
BRCA1 is known to interact with BRCA2 (FANCD1) which in turn interacts with the RAD51
recombinase. RAD51 protein is likely to play a direct role in DNA repair thus completing the
cycle. Not shown: FANCB, which may also be related to BRCA2.
DNA Mismatch Repair (MMR)
Defects in Herditary
Non-Polyposis Colon Cancer
(HNPCC)
Single base mismatches-- misincorporation by DNA polymerase,
missed by proofreading exonuclease.
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Insertion-deletion loops (IDLs)-- caused by polymerase slippage on
repetitive template, gives rise to Microsatallite Instability (MSI).
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E. coli
Methyl-Directed
Mismatch Repair
System
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Eukaryotic Homologs of MutS and MutL
Heterodimers of Eukaryotic MutS & MutL Homologs
*Note: This is yeast nomenclature.
Mlh1 paralogs have different names
in yeast and humans.
Mlh1-Mlh2
Msh2 Msh3
Mlh1-Mlh3
MutLb
Msh2 Msh3 MutSb
MutLa
Rad1-Rad10
Mlh1-Pms1
Mlh1-Pms1
Mlh1-Mlh3
Msh2 Msh3
Msh2 Msh3
Msh2 Msh6
Msh4 Msh5
MutSa
2-4 b
1b
Non-homologous
tail removal in
recombination
intermediates
Insertion/deletion
loop (IDL)
removal
Repair of
base-base mismatches
Promotion of
meiotic crossovers
Model for Eukaryotic Mismatch Repair
HNPCC:
Colon cancer predisposition syndrome, ~5% of all colorectal cancers
Early onset (~40-50 yrs), tumors typically of proximal colon, also with increased
risk for developing tumors of endometrium, ovary, stomach, & small intestine.
Turcott’s syndrome (colorectal tumors & glioblastoma) and Muir-Torre syndrome
(colorectal and skin gland cancers) share genetic features with HNPCC.
Microsatellite instability (MSI) found in mono-, di-, tri-, and tetranucleotide
repeat sequences in tumors taken from HNPCC patients.
MSI linked to defects in any of several MMR genes.
Anti-recombination Effects of MMR Machinery
Observation: MutS, MutL, and to a lesser extent UvrD guard against homeologous
recombination between divergent DNA sequences. Mutations at these loci increase
Homeologous recombination frequencies by 100x to 1000x or more. Similar observations
with MMR mutants in yeast.
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Models to explain rejection of heteroduplex intermediates containing mispairs via MMR
proteins. In this figure, base pair differences between the recipient and donor chromosomes
are indicated by the solid circles. (1) The mismatch correction process itself could lead to
resection of nicked strands and the creation of a single-stranded gap that destroys the
recombination intermediate. (2) hDNA rejection results in the unwinding of the annealed
strands by a helicase that takes its cue from interactions with bound Msh factors. (3) Binding
of MMR factors blocks attempted hDNA formation (Sugawara et al., unpublished).
Anti-homeologous recombination activity of MMR machinery may
be important for:
1. Speciation & evolution. Provides a barrier to inter-species recombination &
thereby reinforces divergent processes.
2. Prevents recombinational deletions of sequence-related genes and thereby
stabilizes divergent gene duplications.
3. These and other factors may contribute to tumor evolution.
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Two steps in recombination in which the Msh2p-Msh3p complex may interact with recombination
intermediates. (Left) Msh2p-Msh3p loads onto DSB sites at recessed ends (1) and/or plays an active role
in scanning hDNA and interacts with loops formed during pairing of homeologous sequences (2), leading
to their rejection from the heteroduplex. (Right) Msh2p-Msh3p binds at the junction of homologous and
nonhomologous DNA allowing for cleavage of unpaired tails by Rad1p-Rad10p (3) (adapted from
reference 17).
Further Reading:
1. Recombinational DNA repair and human disease.
Thompson & Schild (2002) Mutation Research 509, 49-78.
2. Mammalian DNA mismatch repair.
Buermeyer et al. (1999) Annu. Rev. Genet. 33, 533-564.
3. Role of DNA mismatch repair defects in the pathogenesis of human cancer.
Peltomaki (2003) J. Clinical Oncology 21, 1174-1179.