Understanding Structural Mechanisms Of DNA Processing Assemblies

1
Understanding Structural Mechanisms Of DNA
Processing Assemblies

• 1. Modularity multiple domains, separate
  functions
• 2. Multiple contact points XPA
• 3. Modest affinity micromolar contact points

2
RPA Coordinated Activity of Modules
70AB
14/32D/70C
Zn
B
A
C
D
RPA70 RPA32 RPA14
NTD
14
CTD
70NTD
32CTD
3
Interaction of RPA with XPA
RPA Library RPA14/32 RPA14/32DC RPA14/32/70DN RPA7
0DC RPA32C RPA70N RPA70AB
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RPA Modulates Function inMultiple DNA Repair
Pathways
NER
RPA32
BER
RR
5
A Common Mode of Interaction for Different DNA
Repair Proteins
6
RPA Drives Assembly and Commitment to a Specific
Pathway of Repair
NER
RPA32
BER
RR
7
RPA32C- Built for Dynamic Binding Simple Motif
/ Modest Affinity

• Central, flat hydrophobic surface
• Electrostatic complimentarity at either end of
  the binding region

Mer et al., Cell 2000
8
Understanding Structural Mechanisms Of DNA
Processing Assemblies

• 1. Modularity multiple domains, separate
  functions
• 2. Multiple contact points XPA
• 3. Modest affinity micromolar contact points
• 4. Multiple interactions multiple proteins, DNA

9
Using Concepts To Build ModelsUDG A Modular
Damage Recognition Protein

• Multiple contacts with relatively modest
  affinity
• Modularity allows integration of multiple
  functions

10
Continuing to Build View of NER RPA
Interactions with XPG and XPF/ERCC1
1. Recognize damage
2. Unwind duplex
3. Locate lesion
4. Excise 5
RPA
5. Excise 3
3,4,5.

• Cannot all be independent sites on RPA!
• Direct competition for sites, other mechanisms?

11
Understanding Structural Mechanisms Of DNA
Processing Assemblies

• 1. Modularity multiple domains, separate
  functions
• 2. Multiple contact points XPA
• 3. Modest affinity micromolar per contact point
• 4. Multiple interactions multiple proteins, DNA
• Forward progression structural transitions

12
Binding of ssDNA
13
RPA Binds DNA Non-Specifically

• Needs to bind ssDNA regardless of sequence!!

14
Structure Shows Base-Specific Contacts!
OB-Fold
Base-specific contacts

• Needs to bind all ssDNA sequences!!! (X-ray and
  NMR)

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NMR Reveals Solution Same as Crystal

• NMR can be used to study ssDNA binding properties

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NMR Assessment of 3 ssDNA Oligomers

• RPA70AB binds all ssDNA in the same manner

17
ssDNA Binding Site Is Highly Dynamic
S2
S2

• Binding of ssDNA quenches motions

18
Mechanism for Non-Specific Binding
HypothesisThe intrinsically flexible binding
site is able to remodel in response to the
properties of different DNA bases
19
Binding of Proteins
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What Structural Mechanisms Allow Progression Of
SV40 Replication?

• Analyze interactions of RPA with SV40 Large
  T-antigen

21
The SV40 T-ag Origin Recognition Domain Binds to
RPA70AB
Trypsin T-ag
Trypsin T-ag
Trypsin T-ag
Trypsin T-ag
Trypsin T-ag
Trypsin T-ag
Trypsin
Trypsin
Control
Control
Trypsin
Control
RPA-B
RPA-A
RPA-AB
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Affinity of RPA70AB For SV40 T-ag Origin
Recognition Domain Is Modest
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NMR Analysis of Structure and Binding

• Not all residues affected, 100 mM affinity

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Mapping Binding Sites on Structures
T-ag131-259
RPA70AB
Bochkarev et al., 1997
Luo et al., 1996

• Discrete binding sites, modest affinity
• T-ag binds remote from the ssDNA binding site

25
Is T-ag an Allosteric Effector of RPA?
T-ag131-259
RPA70AB
Bochkarev et al., 1997
Luo et al., 1996
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Binding of T-ag Alters Affinity for ssDNA
Normalized RPA Fluorescence
d-CTTCACTTCA T-ag131-259 d-CTTCACTTCA
Molar Ratio of ssDNA

• Pre-loading T-ag increases RPAs affinity for
  ssDNA
• Converse releasing T-ag lowers affinity for
  ssDNA

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Structural Stabilization From Binding

• Tighter binding gives rise to equal or BETTER!!
  spectra in gt40 kDa ternary complex

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What is the mechanism for allostery?
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Mechanism Independent Domains
RPA70AB
RPA70A RPA70B

• A and B domains behave as independent modules
• (in the absence of binding partners)

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Mechanism ssDNA Binding RequiresAlignment of A
and B Domains
ssDNA bound with 5-3 polarity
Different interdomain angles
domains aligned
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Entropic Contribution To Allostery
RPA70AB
Pre-loading T-ag on RPA70AB pre-aligns the A and
B domains
Bochkarev et al., 1997
Lower penalty for loss of entropy higher DNA
affinity
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Model for Dynamic Progression From Unwinding to
Priming
T Ag

• Pol-prim out-competes T-ag for RPA, which causes
  release of ssDNA and hand-off to DNA primase
  step

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Next Step Extend Analysis to Primase

• Identify interaction modules, characterize
  binding

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The Essential Recombination Factor Rad51N Also
Interacts With RPA70AB
51
51
51
51
51
51
RAD52
51
RPA

• Are the structural mechanisms the same as for
  T-ag?

35
Pre-loading Rad51N on RPA70AB Affects the Binding
Affinity for ssDNA
36
But Rad51 Binds Differently to RPA70AB!!
15N RPA70AB
Aihara et al., 1999
Bochkarev et al., 1997
RPA70AB
Rad511-93
Mechanism must be different Allostery versus
direct competition for sites?
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Structural Mechanisms of DNA Processing
Assemblies Key Concepts

• 1. Modularity multiple domains, separate
  functions
• 2. Multiple contact points XPA, T-ag, DNA
  primase
• 3. Modest affinity micromolar per contact point
• 4. Multiple interactions multiple proteins, DNA
• 5. Structural transitions direct competition
  between sites allosteric coupling

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Model for Progression ofDNA Processing Assemblies

• Linked weak, short-lived interactions provide
  high affinity but keep interactions dynamic
• Such dynamic interactions can be invaded and
  rapidly disassembled ? progression