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Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies)

March 6-11, 2003. Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies). Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@ vanderbilt.edu http://structbio.vanderbilt.edu/chazin.

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Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies)

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  1. March 6-11, 2003 Biochemistry 305Structural Mechanisms of the DNA Replication, Recombination and Repair(DNA Processing Assemblies) Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@ vanderbilt.edu http://structbio.vanderbilt.edu/chazin

  2. Processing of DNA is Performed by Multi-protein Assemblies XPC TFIIH XPA T Ag XPF Pol/ Prim SSB XPG SSB Replication Repair 51 51 51 51 51 51 RAD52 51 SSB How do they work? Recombination

  3. DNA Processing Involves Common Steps • Site recognition: know where to start • Helicase: unwind the DNA duplexssDNA • SSB: protect and organize ssDNA • Nuclease: cut ssDNA • Polymerase: synthesize new ssDNA • Ligase: sew ssDNA strands together • Assemblies needed to perform multiple steps

  4. T Ag T Ag SSB SSB SSB T Ag T Ag T Ag T Ag T Ag T Ag T Ag T Ag T Ag T Ag T Ag Pol/ Prim Pol/ Prim T Ag T Ag T Ag T Ag SSB DNA Processing is Dynamic T Ag SV40 Replication • Structural snapshots at different time points (X-ray, EM) • Need to characterize dynamic progression (NMR)

  5. Mechanism of DNA ProcessingPreformed Assemblies? 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 SSB 3,4,5…. • How does progression occur? • Reorganization of a static complex?

  6. Mechanism of DNA ProcessingDynamic Assembly? 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 SSB 3,4,5…. Progression through the multiple steps by dynamic asembly/disassembly of sub-complexes

  7. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 Multiple Proteins/Multiple Steps 1. Recognize damage 2. Unwind duplex 3. Locate lesion 4. Excise 5’ SSB Nucleotide Excision Repair 5. Excise 3’ 3,4,5…. What enables progress through the multiple steps?

  8. Structural Approach To MechanismIntegrated use of NMR, computation and crystallography NMR Computation Crystallography Biophysical Analysis Expression/Mutations RPA-A RPA-B Fluorescence Intensity RPA-AB Ratio of T-ag/RPA

  9. Where to Start?Select the Common Element (SSB) XPC TFIIH XPA XPF T Ag Pol/ Prim SSB XPG SSB Replication Repair 51 51 51 51 51 51 RAD52 51 SSB Recombination

  10. The Eukaryotic SSB isReplication Protein A (RPA) XPC TFIIH XPA XPF T Ag Pol/ Prim RPA XPG RPA Replication Repair 51 51 51 51 51 51 RAD52 51 RPA Recombination

  11. Replication Protein A (RPA) Structure OB-Fold Winged HLH Binds ssDNA Binds proteins • Domains defined by limited proteolysis • Biochemical functions of domains assigned

  12. 3D Structures of RPA Domains X-Ray 14/32D/70C 70AB NMR Zn B A C D RPA70 RPA32 RPA14 NTD 14 CTD 32CTD 70NTD quaternary structure?

  13. Refresher on NMR 15N- 1H HSQC Spectrum R R 15N - Ca- CO - - -15N - Ca H H 15N Frequency 1H Frequency • One peak for each residue • Each peak can be assigned to a single site in the protein

  14. C RPA32 P RPA14 NMR Analysis Of Quaternary Structure 15N RPA32/14 40 173 • Flexibly linked domains: RPA32C is structurally independent of the core of RPA32/14

  15. P RPA: Coordinated Activity of Modules 14/32D/70C 70AB Zn B A C D RPA70 RPA32 RPA14 NTD 14 CTD 70NTD 32CTD

  16. Understanding Structural Mechanisms Of DNA Processing Assemblies 1. Modularity: multiple domains, separate functions

  17. Interaction of RPA with Damage Recognition Protein XPA Nucleotide Excision Repair (NER) Assembly XPC TFIIH XPA XPF XPG RPA Develop tools to address the structural basis of RPA action Mer et al., Cell 2000

  18. Proteolysis Target Immobilized RPA Wash Elute Method to Identify Sites of InteractionMatrix of RPA Affinity Chromatography Columns RPA Library RPA14/32 RPA14/32DC RPA14/32/70DN RPA70DC RPA32C RPA70N RPA70AB Mass Spec

  19. Results Using RPA14/32 as Bait 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

  20. RPA32C Binds XPA N-Terminal Region Control 14/32 14/D32 XPA1-98 FT W1 W2 E FT W1 W2 E FT W1 W2 E E

  21. Proteolysis XPA Immobilized RPA Wash Elute XPAN - RPA32C XPAC - RPA70N Interaction of RPA with XPA RPA Library RPA14/32 RPA14/32DC RPA14/32/70DN RPA70DC RPA32C RPA70N RPA70AB

  22. Understanding Structural Mechanisms of DNA Processing Assemblies • Modularity: multiple domains, separate functions 2. Multiple contact points: XPA

  23. C N Winged Helix-Loop-Helix 3D Structure of RPA32CThe Start - Not The End!! • What is the molecular basis for interaction?

  24. Quantifying RPA32C-XPAN InteractionNMR Titration: 15N-RPA32C + XPAN RPA32C RPA32C + XPA 1-98 R R 15N - Ca- CO - - -15N - Ca H H • Determine affinity (20 mM ) • Map the binding site • Only 19 residues affected  discrete binding site Mer et al., Cell 2000

  25. C N Map Binding Site On The Structure RPA32C Discrete Binding Site • Small surface on XPA • Could be a helical element from XPA

  26. Titration of 15N-XPA1-98 + RPA32C Reveals Side Chains in Binding Site O (CH2)n- C - 15NH2 -15NH- Ca- CO - XPA1-98 XPA1-98 + RPA32C MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM

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

  28. RPA Modulates Function inMultiple DNA Repair Pathways NER RPA32 BER RR

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

  30. A Common Mode of Interaction for Different DNA Repair Proteins UDG79-88 RAD257-274 XPA29-46

  31. RPA Drives Assembly RPA32 UDG

  32. RPA Drives Assembly and Commitment to a Specific Pathway of Repair NER RPA32 BER RR

  33. What Drives RPA32C Interactions?Structure of UDG Peptide Complex C N N RPA32C-UDG RPA32C

  34. UDG Peptide RPA32C Protein Surface Flat Hydrophobic Contact Region

  35. RPA32C UDG Peptide Protein Surface Electrostatic Complimentarity

  36. 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

  37. Understanding Structural Mechanisms Of DNA Processing Assemblies 1. Modularity: multiple domains, separate functions 2. Multiple contact points: XPA 3. Modest affinity: micromolar contact points

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