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

1. 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

2. XPC TFIIH XPA XPF XPG RPA 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

3. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 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

4. Proteolysis XPA Wash Elute Mass Spec Identification Probing RPA/XPA Interactions RPA14/32 Affinity

5. 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 FT E FT E XPA1-273 SDS-PAGE

6. 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

7. XPA P Isolate the RPA32 C-terminal Domain to Determine its Function 40 173 RPA32 RPA14 RPA32C Produce RPA32 C-terminal domain (RPA32C)

8. C N RPA32C NMR StructureThe Starting Point! Winged Helix-Loop-Helix

9. Use NMR to Define XPA Binding Site15N-RPA32C + Unlabeled XPA1-98 RPA32C RPA32C + XPA 1-98 R R 15N - Ca- CO - - -15N - Ca H H • Only 19 residues affected • Discrete binding site • Exchange broadening • Kd > 1 mM

10. C N 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

11. Use NMR to Define RPA-Binding SiteTitration of 15N-XPA1-98 + RPA32C O (CH2)n- C - 15NH2 -15NH- Ca- CO - XPA1-98 XPA1-98 + RPA32C MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM

12. XPA Peptide Induces Same Shifts in RPA32C as Intact N-terminal Domain • Same residues • Same binding site • Slow exchange • Kd < 1 mM XPA1-98 XPA29-46

13. Predict Binding Sites in Other DNA Damage Recognition Proteins ERKRQRALMLRQARLAAR RIQRNKAAALLRLAAR RKLRQKQLQQQFRERMEK NER XPA29-46 UDG79-88 RAD257-274 BER RR Patterns But Not Homology!!!

14. NMR Shows Binding of DNA Damage Recognition Proteins is Identical UDG79-88 RAD257-274 XPA29-46

15. RPA Function From Structural Analysis Regulator of DNA Repair Pathways NER RPA32 BER RR

16. Molecular Basis for RPA32C InteractionsStructure of UDG Peptide Complex C N N RPA32C-UDG RPA32C

17. 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?

18. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 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?

19. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 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

20. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 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

21. 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