mechanisms of accuracy in homologous recombinationtlusty/talks/accuracyrec2008.pdf · mechanisms of...
TRANSCRIPT
![Page 1: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/1.jpg)
Mechanisms of Accuracy in Homologous Recombination
• Recognition processes in homologous recombination
in the presence of competition and noise.
• Mechanism 1: Kinetic Proofreading.
• Mechanism 2: Conformational Proofreading.
• Conformational changes (induced fit) may enhance recognition.
• Possible tests?
Institute Curie, April 2008
![Page 2: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/2.jpg)
RecA-mediated Homologous Recombination
• Essential for genome integrity via repair machinery.
• Molecular engine of genetic diversity via crossover and sex
(horizontal transfer).
• Key role in evolution: control of speciation and genetic isolation.
• Proteins mediate homologous recombination.
• RecA superfamily: RecA (E.coli), Rad51 (yeast), hRad51 (human).
• Structure and function conserved from bacteria to human.
![Page 3: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/3.jpg)
Molecular recognition in Homologous Recombination
RecA Polymerization
Homologous search
Strand exchange
50-60%
• Recombination is a three-stage process:
(1) RecA is polymerized along ssDNA.
• RecA extends ssDNA by 50-60% - conserved.
(2) Homologous search: recognition of homologous DNA
sequence within many lookalikes.
• Homologous search may occur even without ATP
hydrolysis.
(3) Formation of synapse and strand exchange.
![Page 4: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/4.jpg)
Challenge of molecular recognition
• Noisy, crowded milieu.
• Recognizer and target fluctuate.
• Many competing lookalikes.
• Relatively weak recognition interactions.
• Challenge:
How to find and identify targets ?
David Goodsell
![Page 5: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/5.jpg)
How to enhance recognition during recombination?
• The recognition problem is twofold:
• Global-scale: How to approach the target?
• Parallel search (finds target within 200,000
competitors in 15 minutes)
• Local-scale: How to recognize the homologous sequence?
• Two suggested mechanisms:
– Kinetic Proofreading (Libchaber, Bar-Ziv, Stavans, Sagi & TT, 2002-6)
– Conformational Proofreading (Savir & TT, 2007-8)David Goodsell
(Dorfman, Fulconis, Dutreix,Viovy, PRL 2004)
![Page 6: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/6.jpg)
1st mechanism: Kinetic Proofreading
• Kinetic Proofreading (Hopfield, Ninio, 1974-5) = Energy consuming
mechanism for sensitivity amplification in biological processes.
• Allows discrimination between two similar targets (~ free energies)
with an error << naive thermodynamic bound ~ exp(-ΔF).
• Intermediate irreversible stages (coupling to energy source).
TargetsRight product
0 1 Wrong product2Right product
Wrong product
+
+
![Page 7: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/7.jpg)
Hint: Kinetic Proofreading in filament formation
• RecA binding-unbinding dynamics:
Polymerization/depolymerization (ATP).
• Detects minute sequence differences.
0
1
2
final
[ TT, Bar-Ziv, Libchaber PNAS 2002, PRL 2004 ]
![Page 8: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/8.jpg)
Kinetic Proofreading in Recombination
• Experimental hints (Sagi, Stavans & TT, NAR 2006):
• Homology recognition
• FRET measurements of exchange fraction
with heterologous sequences.
• Dependence on the number of
mismathches and their location.
mismatches
![Page 9: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/9.jpg)
Strand exchange depends strongly on location and distribution of mismatches
• Small fraction of mismatches can reduce
significantly the exchange (even << 10%).
• Exchange is directional: more vulnerable to
mismatches close to the 3’ end.
• Contiguous mismatches have stronger effect.
mismatch
![Page 10: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/10.jpg)
Model: Kinetic Proofreading Cascade
• Sequential check and exchange.
• Cascade of N irreversible stages.
• Exchange can abort at each stage.
• Mismatches reduce forward rate.
• Multiplicative effect of mismatches.
→ Exponential decay of exchange 1 1t j j j j j j jp p p pα α β− −∂ = − + −
1
0
success = jN
j j j
pp
αα β
−=+∏
fraction ~ exp( )Lμ− µ – mismatch probability, L – length
![Page 11: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/11.jpg)
Experimental evidence and hints
• Minimum efficient processing segment (MEPS) of 20-30 bp.
• Exponential dependence of successful exchange in vivo
(Vulic Tadei Radman, PNAS 1997; Majewski Zawadzki Pickerill Cohan Dowson, J Bact 2000).
• Such sensitivity sets a well-defined genetic barrier between species.
MEPS ~ 20-30 bp. Red
uctio
n of
exc
hang
e
![Page 12: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/12.jpg)
Role of DNA extension in homologous search?
• Costs significant deformation energy,
3-4 kBT per base-pair (kBT = 0.6 kcal/mol).
• Structural reason: exposing bases?
• Increasing effective target size?(Klapstein Chou Bruinsma 2004)
• Conformational Proofreading?
mechanism to detect DNA in large pool of similar targets, using conformation changes.
![Page 13: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/13.jpg)
Why induced fit?
• Induced fit: Recognizers change their shape upon binding. • Can molecular recognition gain from induced fit?
• Quality measures: specificity = [Right]/[Wrong] =• What is the optimal recognition strategy?
Lock-and-key or induced fit ?
WW
W
WR
Induced fit(Koshland 1958)
?
?
![Page 14: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/14.jpg)
Conformational Proofreading:When off-target is right on
• Structural mismatch reduces Right, but also reduces Wrong even more.
• Result: Enhancement of specificity and other quality measures.
• Optimal specificity at finite mismatch.
• Quantitative example: Homologous Recombination.
recognizersize
RightWrong
Specificity
binding
• Induced fit enhances recognition. • Optimal recognizer is off-target• Not lock-and-key.
Optimal
recognizersize
![Page 15: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/15.jpg)
Molecular recognition as a decision problem
• Natural measure for the performance of molecular recognition .
• Each possible binding event has a cost/benefit of identification and
misidentification.
• Cost = Cost(event)×Prob(event) = correct + miss + false-alarm.
• Cost depends on structural parameters and can be optimized.
No Bind
Bind
Noise
DecisionUnit
Right, Wrong WrongRightTarget
Decision
False Alarm CWBCorrect, CRBBind
Correct, CWNMiss, CRNNo Bind
![Page 16: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/16.jpg)
Cost depends on structural parameters
N base pairs
m mismatches
Extension energy
ΔGext
Gain from correct bp: specific interactions
ΔGs
Gain form incorrect bp: nonspecific interactions
ΔGns
( , ) ( , , , ),binding ext s nsP N m F N m G G G= Δ Δ Δ
• Binding probability for N base pairs and m mismatches :
ssDNA+RecA filament
dsDNA
( , )1 ( · ( )· · )
1binding
ext s nsP N m
exp N G N m G m G=
+ Δ − − Δ − Δ
![Page 17: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/17.jpg)
Cost balances Right and Wrong binding
• Tolerance t measures relative cost of error.
• t increases → system less tolerant to errors
• Cost depends on structural
parameters and binding energetics:
(hom) ( hom)binding bindingCost P t P non= − + × −
Maximize Right detection + Minimize Wrong detection
extGΔ extension
sGΔ specific interactionsnsGΔ nonspecific interactions
mis s nsG G GΔΔ = Δ − Δ
![Page 18: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/18.jpg)
Optimal extension minimizes detection cost
• Minimization of the cost reveals an optimal extension (t = 1):
ext
s
GG
ΔΔ
2ext s mis
mG G GN
Δ = Δ − ΔΔ
ns
s
GG
ΔΔ
1
12mN
−
Unstable
complex
NonspecificV
Specific
1
• Optimal extension energy ~ Specific binding energy.
![Page 19: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/19.jpg)
Extension by a factor of
~ Constant force (Bustamante et al.,2000):stretchGΔ~ 4 5 BsG k T−Δ
~ 1.5 3.5s n BsG G k TΔ − −Δ
χ
Interfacial energy
(Cizeau Viovy, 1997).
~ 3.6 (at 1.7)Bint k TG χ =Δ
Structural parameters are measured
(Xiao Lee Singleton, 2006)
(Malkov Camerini-Otero, 1998)
![Page 20: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/20.jpg)
Cost exhibits minimum at optimal extension
, 13, 3 ,1 mis BN G T tm k= = Δ = =ΔOne RecA monomer, one mismatch, symmetric:
• Well-defined valley ~ 50-60%
![Page 21: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/21.jpg)
Analytic approximation for the cost
linear with good approximation
2
4 ( )· 2[ 1 1]2 ·
int s misstr
int str
mG G GN G NG N G
χΔ Δ − ΔΔΔ
= + −Δ Δ
2· ( 1) ( 1)ext str intG N G Gχ χΔ = Δ − + Δ −
interfacial interaction
stretching interactionspecific interactions
destabilization due to mismatch
1 3[ 1 (10 ) 1]6N m
N Nχ ≅ + − −⇒
(Fulconis Dutreix Viovy, 2005)
![Page 22: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/22.jpg)
Conformational and Kinetic Proofreading
Kinetic Proofreading:Time delay (additional steps)
Energy-consuming non-equilibrium.
Conformational Proofreading:Spatial mismatch. Quasi-equilibrium.
• Kinetic and Conformational proofreading use the same generalstrategy: Reduce production of both Right and Wrong, but .reduction of Wrong product is larger and specificity improves.
• Recent evidence in recombination.
Sagi, Tlusty, Stavans (2006 ) NAR
0 1 2 final
0 0
Savir, Tlusty (2007-8 ) PLoS ONE, IEEE…
![Page 23: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/23.jpg)
Possible experimental tests?
Conformational Proofreading:
• Binding curves as a function of
controlled extension.
• Predicting increase of binding
fraction with extension.
• But optimal cost at zero extension.
homologous Non-homologous
1 1.5
1 1.5
%hom %non-hom−
% b
ound
Extension
hom
Non-hom
Extension
(Fulconis, Mine, Bancaud, Dutreix, Viovy
EMBO J 2006 )
![Page 24: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/24.jpg)
Possible experimental tests?
Kinetic Proofreading:
• Single molecule measurements of sequential
and directional processes.
• For example, asymmetric correlation etc.
(Fulconis, Mine, Bancaud, Dutreix, Viovy
EMBO J 2006 )
Typical KPR dynamics
![Page 25: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/25.jpg)
Summary and outlook
• Suggestion: Homologous recombination combines
Kinetic Proofreading + Conformational Proofreading.
• Indirect experimental evidence. Direct evidence?
• Conformational Proofreading: conformational changes (induced fit)
may enhance the quality of molecular recognition.
• Analogy between molecular recognition and decision problem may
explain the extreme DNA extension by 50-60%.
• Outlook: Application of Conformational Proofreading to other systems:
tRNA, transcription, enzymes… (in progress).
[ Savir & Tlusty, PLoS ONE 2007; IEEE J Signal Processing 2008; RecA – submitted ]
![Page 26: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/26.jpg)
Optimal extension is not sensitive to tolerance
3, 1 1 5 3, . ,s B mis BN m G k k TT G= = Δ = ΔΔ =
mism Gct e ΔΔ≅
• t increases → system less tolerant to errors
![Page 27: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/27.jpg)
Cost balances correct and incorrect binding 1 .
1 ( · · ) 1 ( · · · )ext s ext s mis
tCostexp N G N G exp N G N G m G
−= +
+ Δ − Δ + Δ − Δ + ΔΔ
Maximize correct detection + Minimize incorrect detection
extGΔ extension
sGΔ specific interactionsnsGΔ nonspecific interactions
mis s nsG G GΔΔ = Δ − Δ
• t – Tolerance of the System
correct
incorrect
· (hom)· (hom)· (non-hom)· (non-hom)
h f
h f
c p pc p p
t = −
Cost Occurrence functionality
t increases the system is less tolerant to errors
![Page 28: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/28.jpg)
Cost exhibits minimum at optimal extension
, 13, 3 ,1 mis BN G T tm k= = Δ = =Δ• One RecA monomer, one mismatch, symmetric:
![Page 29: Mechanisms of Accuracy in Homologous Recombinationtlusty/talks/AccuracyRec2008.pdf · Mechanisms of Accuracy in Homologous Recombination ... genetic diversity via crossover and sex](https://reader035.vdocuments.net/reader035/viewer/2022070722/5ee3c80cad6a402d666d67b0/html5/thumbnails/29.jpg)
Homologues Recombination
• Genome repair• Generating genetic diversity
Bugreev et al, Nature Structural & Molecular Biology 14, 746 - 753 (2007)