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Understanding Structural Mechanisms Of DNA Processing Assemblies

Explore the modularity and multiple contact points in DNA processing assemblies, including RPA and XPA interactions. Understand the modest affinity and multiple protein interactions for efficient DNA repair pathways. Discover the structural transitions and binding mechanisms involved in DNA repair processes.

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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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