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Understanding Structural Mechanisms Of DNA Processing Assemblies

Understanding Structural Mechanisms Of DNA Processing Assemblies. 1. Modularity: multiple domains, separate functions 2. Multiple contact points: XPA 3. Modest affinity: micromolar contact points. P. RPA: Coordinated Activity of Modules. 14/32D/70C. 70AB. Zn. B. A. C. D. RPA70

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Understanding Structural Mechanisms Of DNA Processing Assemblies

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

  3. 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

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

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

  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. UDG Using Concepts To Build ModelsUDG: A Modular Damage Recognition Protein • Multiple contacts with relatively modest affinity • Modularity allows integration of multiple functions

  10. 1 XPC 3 2 TFIIH 5 XPA XPF XPG 4 Continuing to Build View of NERRPA 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)

  15. NMR Reveals Solution Same as Crystal • NMR can be used to study ssDNA binding properties

  16. 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

  20. RPA RPA RPA T Ag T Ag T Ag T Ag T Ag T Ag Pol/ Prim T Ag Pol/ Prim T Ag T Ag RPA 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

  22. Normalized RPA Fluorescence Molar Ratio of T-ag Affinity of RPA70AB For SV40 T-ag Origin Recognition Domain Is Modest

  23. 15N T-ag131-259 15N RPA70AB 15N 1H NMR Analysis of Structure and Binding • Not all residues affected, ~100 mM affinity

  24. 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

  26. 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 RPA’s affinity for ssDNA • Converse: releasing T-ag lowers affinity for ssDNA

  27. RPA70AB/ssDNA+ T-ag RPA70AB/T-ag+ ssDNA 15N Structural Stabilization From Binding • Tighter binding gives rise to equal or BETTER!! spectra in >40 kDa ternary complex

  28. What is the mechanism for allostery?

  29. Mechanism: Independent Domains RPA70AB RPA70A + RPA70B • A and B domains behave as independent modules • (in the absence of binding partners)

  30. Mechanism: ssDNA Binding RequiresAlignment of A and B Domains ssDNAbound with 5’-3’ polarity Different interdomain angles domains aligned

  31. 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

  32. T Ag T Ag RPA RPA RPA T Ag 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 T Ag T Ag RPA RPA 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

  33. RPA RPA RPA T Ag T Ag T Ag T Ag T Ag T Ag Pol/ Prim T Ag Pol/ Prim T Ag T Ag RPA Next Step: Extend Analysis to Primase • Identify interaction modules, characterize binding

  34. 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. d-CTTCACTTCA + Rad51N Normalized RPA Fluorescence Molar Ratio of ssDNA 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?

  37. 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

  38. 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

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