A Case Study RPA: A Multi-domain, Multi-subunit Protein

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A Case StudyRPA A Multi-domain, Multi-subunit
Protein
RPA70
Zn
RPA32
P
Binds ssDNA
Binds proteins
RPA14

• Quaternary structure unknown, partial function
• Delineation of domains by limited proteolysis

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Protein Interactions in Biology RPA/XPA in
Nucleotide Excision Repair

• DNA damage must be repaired
• Malfuction of repair leads to cancer
• Goal Understand repair to make anticancer drug

RPA is an essential component of the NER pathway
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The NER Complex is a Protein Machine
1. Recognize damage
2. Unwind duplex
3. Locate lesion
4. Excise 5
RPA
5. Excise 3
3,4,5.
RPA is required at multiple steps
Machines perform multiple tasks
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Probing RPA/XPA Interactions
RPA14/32 Affinity
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Binding of XPA by RPA14/32
Control
14/32
14/D32
Mass Spec all bound fragments have XPA1-98
C-terminus of RPA32 required for binding XPA
FT
E
E
FT
E
FT
XPA1-273
SDS-PAGE
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XPA N-Terminal Domain Binds RPA
Control
14/32
14/D32
XPA1-98
FT W1 W2 E
FT W1 W2 E
FT W1 W2 E
E
SDS-PAGE
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Isolate the RPA32 C-terminal Domain to Determine
its Function
173
40
RPA32
RPA14
RPA32C
Produce RPA32 C-terminal domain (RPA32C)
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RPA32C NMR StructureThe Starting Point!
Winged Helix-Loop-Helix
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Use NMR to Define XPA Binding Site15N-RPA32C
Unlabeled XPA1-98

• Only 19 residues affected
• Discrete binding site
• Exchange broadening
• Kd gt 1 mM

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Perturbations in NMR Spectrum Mapped onto RPA32C
Structure

• Winged Helix-Loop-Helix
• Discrete surface
• Different from HLH surface for dsDNA
• RPA32C does not bind dsDNA

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Use NMR to Define RPA-Binding Site Titration of
15N-XPA1-98 RPA32C
MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSA
TAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM
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XPA Peptide Induces Same Shifts in RPA32C as
Intact N-terminal Domain

• Same residues
• Same binding site
• Slow exchange
• Kd lt 1 mM

XPA1-98
XPA29-46
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Predict Binding Sites in Other DNA Damage
Recognition Proteins
E R K R Q R A L M L R Q A R L A A R R I Q R N K A
A A L L R L A A R R K L R Q K Q L Q Q Q F R E R M
E K
NER
XPA29-46 UDG79-88 RAD257-274
BER
RR
Patterns But Not Homology!!!
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NMR Shows Binding of DNA Damage Recognition
Proteins is Identical
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RPA Function From Structural Analysis Regulator
of DNA Repair Pathways
NER
RPA32
BER
RR
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Molecular Basis for RPA32C InteractionsStructure
of UDG Peptide Complex
C
N
N
RPA32C-UDG
RPA32C
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Detailed Insights by Identifying Critical
Interactions in the Complex

• Structure reveals why 3 different DNA damage
  recognition proteins bind to RPA32
• How to generate specificity in drug targeting?

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How Does the NER Machine Function?
1. Recognize damage
2. Unwind duplex
3. Locate lesion
4. Excise 5
RPA
5. Excise 3
3,4,5.
RPA is required at multiple steps
Structural model for the NER machine must provide
for progress through the multiple steps of NER?
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Is the NER Complex Pre-formed?
1. Recognize damage
2. Unwind duplex
3. Locate lesion
4. Excise 5
RPA
5. Excise 3
3,4,5.
RPA is required at multiple steps
Progression through the multiple steps of NER
by reorganization of a static complex
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Is the NER Complex a Dynamic Assembly?
1. Recognize damage
2. Unwind duplex
3. Locate lesion
4. Excise 5
RPA
5. Excise 3
3,4,5.
RPA is required at multiple steps
Progression through the multiple steps of NER by
dynamic asembly/disassembly of the complex
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NMR is a Powerful Means to Study Dynamic
Biomolecular Systems
1
XPC
3
2
TFIIH
5
XPA
XPF
XPG
4
RPA
3,4,5.

• Progression by multiple short-lived interactions
• Modularity facilitates dynamic assembly