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E-mail: Walter.Chazin@ vanderbilt.edu
March 6-11, 2003 Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies) Walter Chazin 5140 BIOSCI/MRBIII vanderbilt.edu
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Processing of DNA is Performed by Multi-protein Assemblies
XPC TFIIH XPA T Ag XPF Pol/ Prim SSB XPG SSB Replication Repair Made this one is animated. Arrow gives you a lead in to next slide. 51 51 51 51 51 51 RAD52 51 SSB How do they work? Recombination
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DNA Processing Involves Common Steps
Site recognition: know where to start Helicase: unwind the DNA duplexssDNA SSB: protect and organize ssDNA Nuclease: cut ssDNA Polymerase: synthesize new ssDNA Ligase: sew ssDNA strands together Assemblies needed to perform multiple steps
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DNA Processing is Dynamic
T Ag Pol/ Prim T Ag SSB T Ag SSB T Ag SSB Pol/ Prim T Ag T Ag SV40 Replication Structural snapshots at different time points (X-ray, EM) Need to characterize dynamic progression (NMR)
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Mechanism of DNA Processing Preformed Assemblies?
1 XPC 3 TFIIH 2 5 XPA XPF XPG 4 SSB 3,4,5…. How does progression occur? Reorganization of a static complex?
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Mechanism of DNA Processing Dynamic Assembly?
1 XPC 3 TFIIH 2 5 XPA XPF XPG 4 SSB 3,4,5…. Progression through the multiple steps by dynamic asembly/disassembly of sub-complexes
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Multiple Proteins/Multiple Steps
XPC 1 1. Recognize damage XPF 5 XPA 3 TFIIH 2 2. Unwind duplex XPG 4 3. Locate lesion 4. Excise 5’ SSB Nucleotide Excision Repair 5. Excise 3’ 3,4,5…. What enables progress through the multiple steps?
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Structural Approach To Mechanism Integrated use of NMR, computation and crystallography
Biophysical Analysis Expression/Mutations RPA-A RPA-B Fluorescence Intensity RPA-AB Ratio of T-ag/RPA
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Where to Start? Select the Common Element (SSB)
XPC TFIIH XPA T Ag XPF Pol/ Prim SSB XPG SSB Replication Repair Made this one is animated. Arrow gives you a lead in to next slide. 51 51 51 51 51 51 RAD52 51 SSB Recombination
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The Eukaryotic SSB is Replication Protein A (RPA)
XPC TFIIH XPA T Ag XPF Pol/ Prim RPA XPG RPA Replication Repair Made this one is animated. Arrow gives you a lead in to next slide. 51 51 51 51 51 51 RAD52 51 RPA Recombination
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Replication Protein A (RPA) Structure
OB-Fold Winged HLH Binds ssDNA Binds proteins Domains defined by limited proteolysis Biochemical functions of domains assigned
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3D Structures of RPA Domains
X-Ray 14/32D/70C 70AB NMR Zn B A C D RPA70 RPA32 RPA14 NTD 14 CTD 32CTD quaternary structure? 70NTD
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Refresher on NMR 15N- 1H HSQC Spectrum R 15N - Ca- CO - - -15N - Ca H
15N Frequency 1H Frequency 15N - Ca- CO N - Ca H R 15N- 1H HSQC Spectrum One peak for each residue Each peak can be assigned to a single site in the protein
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NMR Analysis Of Quaternary Structure
15N RPA32/14 40 173 RPA32 RPA14 P C Flexibly linked domains: RPA32C is structurally independent of the core of RPA32/14 Check out the animation. Anders came up w/the idea. I think it works better than what we had discussed.
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RPA: Coordinated Activity of Modules
14/32D/70C 70AB P Zn B A C D RPA70 RPA32 RPA14 NTD 14 CTD 70NTD 32CTD
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Understanding Structural Mechanisms Of DNA Processing Assemblies
1. Modularity: multiple domains, separate functions
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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
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Method to Identify Sites of Interaction Matrix of RPA Affinity Chromatography Columns
Proteolysis Target RPA Library RPA14/32 RPA14/32DC RPA14/32/70DN RPA70DC RPA32C RPA70N RPA70AB Immobilized RPA Elute Wash Mass Spec
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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
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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
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Interaction of RPA with XPA
Proteolysis XPA RPA Library RPA14/32 RPA14/32DC RPA14/32/70DN RPA70DC RPA32C RPA70N RPA70AB Immobilized RPA Elute Wash XPAN - RPA32C XPAC - RPA70N
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Understanding Structural Mechanisms of DNA Processing Assemblies
Modularity: multiple domains, separate functions 2. Multiple contact points: XPA
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3D Structure of RPA32C The Start - Not The End!!
Winged Helix-Loop-Helix What is the molecular basis for interaction?
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Quantifying RPA32C-XPAN Interaction NMR Titration: 15N-RPA32C + XPAN
15N - Ca- CO N - Ca H R Determine affinity (20 mM ) Map the binding site Only 19 residues affected discrete binding site RPA32C RPA32C + XPA 1-98 Mer et al., Cell 2000
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Map Binding Site On The Structure
RPA32C Discrete Binding Site Small surface on XPA Could be a helical element from XPA
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Titration of 15N-XPA1-98 + RPA32C Reveals Side Chains in Binding Site
- 15NH - Ca- CO - (CH2)n- C - 15NH2 O MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM This is animated XPA1-98 XPA RPA32C
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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
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RPA Modulates Function in Multiple DNA Repair Pathways
NER RPA32 BER Summary of Georges’ half of the talk. This is where you come back to NER, BER, RR connection. Craig “unified theory” picture. Isn’t it pretty? You owe him. RR
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Predict Binding Sites in Other DNA Repair 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 RAD BER RR Patterns But Not Homology!!!
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A Common Mode of Interaction for Different DNA Repair Proteins
XPA29-46 UDG79-88 RAD
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RPA Drives Assembly RPA32 UDG
Summary of Georges’ half of the talk. This is where you come back to NER, BER, RR connection. Craig “unified theory” picture. Isn’t it pretty? You owe him.
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RPA Drives Assembly and Commitment to a Specific Pathway of Repair
NER RPA32 BER Summary of Georges’ half of the talk. This is where you come back to NER, BER, RR connection. Craig “unified theory” picture. Isn’t it pretty? You owe him. RR
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What Drives RPA32C Interactions? Structure of UDG Peptide Complex
RPA32C-UDG RPA32C
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Flat Hydrophobic Contact Region
UDG Peptide RPA32C Protein Surface
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Electrostatic Complimentarity
RPA32C UDG Peptide Protein Surface
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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
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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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