parS
Keeping it together: ! Organizing the bacterial chromosome for division! Ned Wingreen
Chase Broedersz
Princeton University!
Xindan Wang, Joseph Loparo, and David Rudner Harvard Medical School! Yigal Meir
Ben-Gurion University 1
Outline
• The problem of packaging and moving DNA
- Nucleoid Associated Proteins
- The ParABS system
• How does ParB condense DNA?
• Lattice model for the ParB-DNA complex
- Localizing the complex
- Roadblocks and loops
- Gene silencing
• Open questions
• Conclusions
2
Packaging DNA is a challenge
E. coli
Kavenoff and Bowen, Chromosome (1976) 3
Chromosomal organization: Nucleoid-associated proteins Bridging and looping
Bending and coiling
Luijsterburg et al. Journal of Structural Biology (2006)
4
ParABS system: Chromosome segregation in Caulobacter crescentus ParB
ParA
1 μm Ptacin et al. Nature Cell Biology (2010)! Shebelut et al. PNAS (2010) !
•How are ~1000 ParB proteins localized on the DNA?! •How do ParBs interact and what is the structure of the ParB-DNA complex?! •What controls formation of the complex?! !
5
ParB clusters around parS sites in Bacillus subtilis Roadblock suppresses spreading
20 kbp’s
Murray et al. Molecular Microbiology (2006)
Breier and Grossman, Molecular Microbiology (2007)
Rodionov et al. Science (1999) !
“spreading” model DNA ParB
parS 6
ParB clusters around parS sites in Bacillus subtilis ParB structure
20 kbp’s Sullivan et al. Cell, 137, 685-696 (2009)
Murray et al. Molecular Microbiology (2006)
Leonard et al. Molecular Microbiology (2004)
“spreading” model DNA ParB
parS 7
Lattice model for DNA-binding proteins
Protein
A
A
Searching for a minimal model for ParB B E C B E D
C & bridging Spreading D or bridging Spreading Spreading & bridging Spreading or bridging model Bridging interactions Bridging model Spreading or bridging Bridging interactions interactionsinteractions A Non-interacting dimers B Spreading interactions Cinteractions Bridginginteractions interactions
Non-interacting dimers Spreading interactions Non-interacting dimers Spreading interactions Dimer model Spreading model
D Spreading or bridging E
D
interactions D
0
10
Spreading or bridging Spreading or bridging Bridging interactions Bridging interactions interactions interactions
F 0
Dimer
10
Dimer
Bridging
Bridging
Spreading 10
Probability p(n)
C
Probability p(n)
C
Spreading & bridging interactions
Spreading
10
Spreading or bridging
Spreading or bridging
10
Spreading & bridging
10
Cluster size distribution
10
10
0
20
10
10
0
10
20
30
40
50
60
Cluster size
70
40
60
Cluster size
80
80
90
100
100
9
eading interactions
int
er
g o ac r br tio idg ns in g
Spreading & bridging model condenses DNA E Spreading & bridging interactions Spreading & Bridging model
A
0
10
Dimer Bridging Spreading
preading or bridging interactions Probability p(n)
10
Spreading or bridging Spreading & bridging
10
10
10
0
10
20
30
40
50
60
Cluster size
70
80
90
100
10
Can a single parS site localize the ParB-DNA complex? A
C parS 1 0.9
Probability
0.8 0.7
Spreading Bridging Spreading or bridging Spreading & bridging
0.6 0.5 0.4 0.3 0.2 0.1 0 0
100
200
300
400
500
Genomic position
Condensation is essential for localization 11
How strong a parS site is needed for localization? Entropic “cost” of localization
B
D parS 1 0.9 0.8
parS
Probability
0.7 0.6 0.5
parS strength 0 1 2 3 4 6
0.4 0.3 0.2 0.1 0 0
200
400
600
Genomic position
In a cell: Mc ~ 1000, N ~ 130,000
∆∊parS < -4.2 kT
800
1000
Roadblock experiment is captured by spreading & bridging model C
roadblock parS 1 0.9
Probability
0.8 0.7
Spreading Bridging Spreading or bridging Spreading & bridging
0.6 0.5 0.4 0.3 0.2 0.1 0 0
100
200
300
400
500
Genomic position
Why doesn’t cluster loop around roadblock?
Statistics of DNA looping
A Scaling of loop number
Universal loop-size distribution
0
10
−1
10
−2
Probability distr.
10
−3
10
−4
10
−5
10
−6
10
α ≃ 0.9
−7
10
0
10
1
2
10
10
3
10
Loop size
B
Projected cluster size
Genomic position
Prediction: ParB overexpression should overcome roadblock ParB cluster
roadblock
D
A
parS 1
0
10
ParB expression level
0.9 0.8
20 40 60 80 100 150 200 250 300
10
Probability
0.7 0.6
10
1
2
10
3
10
10
ParB expression level 0.5 0.4 0.3 0.2
B
0.1 0 0
100
200
300
400
500
600
700
800
900
Projected cluster size
1000
Genomic distance Genomic position
ParB silences genes flanking the parS site in P1 plasmids
Rodionov et al. Science (1999) !
Gene-silencing profiles are captured by the spreading & bridging model DNA exposure 0.9 0.8
DNA exposure DNA exposure
l
E E
1
increasing parS-distance
1
0.9 0.7 0.8 0.6 0.6 0.4 0.4
0.3 0.1 0 0 0.1
0
D D
50
DNA exposure DNA exposure DNA exposure
10
0 0
1010
10
10
tion/ParB expression level 0
0.5
1
sition/ParB expression level
400
450
100
150
300
350
400
450
1 4 6 10 1 1 30 4 4 60 6 6 90 10 10 3030 18060 60 9090 310 410180 180
10
310 310 410 410
1010 1010
1010
0 0
500
F F 5
-1
5
1
3
4
5
6
7
1
3
4
5
6
7
ParB (ng/ g protein)
Collapse of experimental data
0
10
0
10
-1
parS distance
10
0
1
10
10
10
0
0 1010
1
1 1010
1010
ParBexpression expressionlevel/(parS level/(parSdistance) distance) ParB
parS distance 10 10
3
10
ParB expression level/(parS distance)
1.5
0 0
500
ParB (ng/ g protein)
parSdistance distance parS
1010
1.5
350
parS distance
10
1
300
Collapse of numerical data
0
1010
0.5
150
ParB expression level
10
0
100
0.4
ParB expression level
0 0
500
Genomic position
50
Remaining activity Remaining activity
enomic position
0.8 1
0.6 0.4
0.5 0.3
500
increasing parS-distance
0.8 0.6
0.7 0.5
0
1
Remaining activity Remaining activity
C C
Gene activity
0
1
10
10
ParB/(parS distance) 3
3 10 10
10
0
10
1
10
ParB/(parS distance)
17
• Why is parS
required for cluster formation?
Open questions
• How is ParB partitioned between parS sites?
Ptacin et al. Nature Cell Biology (2010)
Sullivan et al. Cell (2009)
18
Localization and condensation of the partitioning protein ParB on the bacterial chromosome
Conclusions
• Bacterial chromosomes/plasmids are organized by DNA-binding proteins
• A minimal “spreading & bridging” model for ParB accounts for:
- Stability and localization of ParB-DNA complexes at parS sites
- Roadblock experiments & Gene silencing
ParB expression level
0.6
50 100
E
1
0.9 0.8
300 400
0.4
1
Remaining activity
0.8
Probability
C
1
DNA exposure
A
0.7 0.6 0.5 0.4 0.3
0.8 0.6 0.4
0.1
0
0 0
500
50
100
150
Genomic position
B
1
0
10
D
0.6 0.4
0.6
10
1
2
10
10
ParB expression level 0.5 0.4
450
0 0
3
4
5
6
7
F
0
parS distance 1 4 6 10 30 60 90
5
180
10
parS distance 10
310 410
0.1
0
1
ParB (ng/ g protein)
0.2
0
0 0
500
10
0.3
!
20 40 60 80 100 150 200 250 300
10
3
10
10
DNA exposure
10
0.7
Probability
Probability
!
0.8
400
0 ParB expression level
0.9
0.8
350
ParB expression level
parS
1
300
Remaining activity
0
0.5 200
1 400
600 1.5
10 800 10
0
1
3
• Open questions
- Mechanism of nucleation at parS sites?
- Partitioning of ParB (role of DNA supercoiling?) 1000
100
300
500
Genomic distance
700
Genomic position/ParB expression level
900
1000
10
10
10
10
ParB expression level/(parS distance)
10
10
0
10
1
10
ParB/(parS distance)
Acknowledgements!
Funding!
Chase Broedersz (Princeton University)!
!
Joseph Loparo, Xindan Wang, and David Rudner (Harvard)! Yigal Meir (Ben Gurion)
19
Number of parS motifs ranges from 1 in B. burgdorferii to 24 in S. coelicolor
ParB2 (Spo0J2) can bind DNA both specifically and nonspecifically
Lin and Grossman, Cell (1998)! Jakimowicz et al., Molecular Microbiology (2002)! Breier and Grossman, Molecular Microbiology (2007) 20
Chase Broedersz
Princeton University!
Xindan Wang, Joseph Loparo, and David Rudner Harvard Medical School! Yigal Meir
Ben-Gurion University 1
Outline
• The problem of packaging and moving DNA
- Nucleoid Associated Proteins
- The ParABS system
• How does ParB condense DNA?
• Lattice model for the ParB-DNA complex
- Localizing the complex
- Roadblocks and loops
- Gene silencing
• Open questions
• Conclusions
2
Packaging DNA is a challenge
E. coli
Kavenoff and Bowen, Chromosome (1976) 3
Chromosomal organization: Nucleoid-associated proteins Bridging and looping
Bending and coiling
Luijsterburg et al. Journal of Structural Biology (2006)
4
ParABS system: Chromosome segregation in Caulobacter crescentus ParB
ParA
1 μm Ptacin et al. Nature Cell Biology (2010)! Shebelut et al. PNAS (2010) !
•How are ~1000 ParB proteins localized on the DNA?! •How do ParBs interact and what is the structure of the ParB-DNA complex?! •What controls formation of the complex?! !
5
ParB clusters around parS sites in Bacillus subtilis Roadblock suppresses spreading
20 kbp’s
Murray et al. Molecular Microbiology (2006)
Breier and Grossman, Molecular Microbiology (2007)
Rodionov et al. Science (1999) !
“spreading” model DNA ParB
parS 6
ParB clusters around parS sites in Bacillus subtilis ParB structure
20 kbp’s Sullivan et al. Cell, 137, 685-696 (2009)
Murray et al. Molecular Microbiology (2006)
Leonard et al. Molecular Microbiology (2004)
“spreading” model DNA ParB
parS 7
Lattice model for DNA-binding proteins
Protein
A
A
Searching for a minimal model for ParB B E C B E D
C & bridging Spreading D or bridging Spreading Spreading & bridging Spreading or bridging model Bridging interactions Bridging model Spreading or bridging Bridging interactions interactionsinteractions A Non-interacting dimers B Spreading interactions Cinteractions Bridginginteractions interactions
Non-interacting dimers Spreading interactions Non-interacting dimers Spreading interactions Dimer model Spreading model
D Spreading or bridging E
D
interactions D
0
10
Spreading or bridging Spreading or bridging Bridging interactions Bridging interactions interactions interactions
F 0
Dimer
10
Dimer
Bridging
Bridging
Spreading 10
Probability p(n)
C
Probability p(n)
C
Spreading & bridging interactions
Spreading
10
Spreading or bridging
Spreading or bridging
10
Spreading & bridging
10
Cluster size distribution
10
10
0
20
10
10
0
10
20
30
40
50
60
Cluster size
70
40
60
Cluster size
80
80
90
100
100
9
eading interactions
int
er
g o ac r br tio idg ns in g
Spreading & bridging model condenses DNA E Spreading & bridging interactions Spreading & Bridging model
A
0
10
Dimer Bridging Spreading
preading or bridging interactions Probability p(n)
10
Spreading or bridging Spreading & bridging
10
10
10
0
10
20
30
40
50
60
Cluster size
70
80
90
100
10
Can a single parS site localize the ParB-DNA complex? A
C parS 1 0.9
Probability
0.8 0.7
Spreading Bridging Spreading or bridging Spreading & bridging
0.6 0.5 0.4 0.3 0.2 0.1 0 0
100
200
300
400
500
Genomic position
Condensation is essential for localization 11
How strong a parS site is needed for localization? Entropic “cost” of localization
B
D parS 1 0.9 0.8
parS
Probability
0.7 0.6 0.5
parS strength 0 1 2 3 4 6
0.4 0.3 0.2 0.1 0 0
200
400
600
Genomic position
In a cell: Mc ~ 1000, N ~ 130,000
∆∊parS < -4.2 kT
800
1000
Roadblock experiment is captured by spreading & bridging model C
roadblock parS 1 0.9
Probability
0.8 0.7
Spreading Bridging Spreading or bridging Spreading & bridging
0.6 0.5 0.4 0.3 0.2 0.1 0 0
100
200
300
400
500
Genomic position
Why doesn’t cluster loop around roadblock?
Statistics of DNA looping
A Scaling of loop number
Universal loop-size distribution
0
10
−1
10
−2
Probability distr.
10
−3
10
−4
10
−5
10
−6
10
α ≃ 0.9
−7
10
0
10
1
2
10
10
3
10
Loop size
B
Projected cluster size
Genomic position
Prediction: ParB overexpression should overcome roadblock ParB cluster
roadblock
D
A
parS 1
0
10
ParB expression level
0.9 0.8
20 40 60 80 100 150 200 250 300
10
Probability
0.7 0.6
10
1
2
10
3
10
10
ParB expression level 0.5 0.4 0.3 0.2
B
0.1 0 0
100
200
300
400
500
600
700
800
900
Projected cluster size
1000
Genomic distance Genomic position
ParB silences genes flanking the parS site in P1 plasmids
Rodionov et al. Science (1999) !
Gene-silencing profiles are captured by the spreading & bridging model DNA exposure 0.9 0.8
DNA exposure DNA exposure
l
E E
1
increasing parS-distance
1
0.9 0.7 0.8 0.6 0.6 0.4 0.4
0.3 0.1 0 0 0.1
0
D D
50
DNA exposure DNA exposure DNA exposure
10
0 0
1010
10
10
tion/ParB expression level 0
0.5
1
sition/ParB expression level
400
450
100
150
300
350
400
450
1 4 6 10 1 1 30 4 4 60 6 6 90 10 10 3030 18060 60 9090 310 410180 180
10
310 310 410 410
1010 1010
1010
0 0
500
F F 5
-1
5
1
3
4
5
6
7
1
3
4
5
6
7
ParB (ng/ g protein)
Collapse of experimental data
0
10
0
10
-1
parS distance
10
0
1
10
10
10
0
0 1010
1
1 1010
1010
ParBexpression expressionlevel/(parS level/(parSdistance) distance) ParB
parS distance 10 10
3
10
ParB expression level/(parS distance)
1.5
0 0
500
ParB (ng/ g protein)
parSdistance distance parS
1010
1.5
350
parS distance
10
1
300
Collapse of numerical data
0
1010
0.5
150
ParB expression level
10
0
100
0.4
ParB expression level
0 0
500
Genomic position
50
Remaining activity Remaining activity
enomic position
0.8 1
0.6 0.4
0.5 0.3
500
increasing parS-distance
0.8 0.6
0.7 0.5
0
1
Remaining activity Remaining activity
C C
Gene activity
0
1
10
10
ParB/(parS distance) 3
3 10 10
10
0
10
1
10
ParB/(parS distance)
17
• Why is parS
required for cluster formation?
Open questions
• How is ParB partitioned between parS sites?
Ptacin et al. Nature Cell Biology (2010)
Sullivan et al. Cell (2009)
18
Localization and condensation of the partitioning protein ParB on the bacterial chromosome
Conclusions
• Bacterial chromosomes/plasmids are organized by DNA-binding proteins
• A minimal “spreading & bridging” model for ParB accounts for:
- Stability and localization of ParB-DNA complexes at parS sites
- Roadblock experiments & Gene silencing
ParB expression level
0.6
50 100
E
1
0.9 0.8
300 400
0.4
1
Remaining activity
0.8
Probability
C
1
DNA exposure
A
0.7 0.6 0.5 0.4 0.3
0.8 0.6 0.4
0.1
0
0 0
500
50
100
150
Genomic position
B
1
0
10
D
0.6 0.4
0.6
10
1
2
10
10
ParB expression level 0.5 0.4
450
0 0
3
4
5
6
7
F
0
parS distance 1 4 6 10 30 60 90
5
180
10
parS distance 10
310 410
0.1
0
1
ParB (ng/ g protein)
0.2
0
0 0
500
10
0.3
!
20 40 60 80 100 150 200 250 300
10
3
10
10
DNA exposure
10
0.7
Probability
Probability
!
0.8
400
0 ParB expression level
0.9
0.8
350
ParB expression level
parS
1
300
Remaining activity
0
0.5 200
1 400
600 1.5
10 800 10
0
1
3
• Open questions
- Mechanism of nucleation at parS sites?
- Partitioning of ParB (role of DNA supercoiling?) 1000
100
300
500
Genomic distance
700
Genomic position/ParB expression level
900
1000
10
10
10
10
ParB expression level/(parS distance)
10
10
0
10
1
10
ParB/(parS distance)
Acknowledgements!
Funding!
Chase Broedersz (Princeton University)!
!
Joseph Loparo, Xindan Wang, and David Rudner (Harvard)! Yigal Meir (Ben Gurion)
19
Number of parS motifs ranges from 1 in B. burgdorferii to 24 in S. coelicolor
ParB2 (Spo0J2) can bind DNA both specifically and nonspecifically
Lin and Grossman, Cell (1998)! Jakimowicz et al., Molecular Microbiology (2002)! Breier and Grossman, Molecular Microbiology (2007) 20
