1. 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

2. 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

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. 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)

5. 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?

6. 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

7. 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?

8. 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

9. 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

10. 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

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
quaternary structure?
70NTD

13. 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

14. 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.

15. RPA: Coordinated Activity of Modules
14/32D/70C
70AB P 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. 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

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. 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

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

23. 3D Structure of RPA32C The Start - Not The End!!
Winged Helix-Loop-Helix
What is the molecular basis for interaction?

24. 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

25. 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
- 15NH - Ca- CO -
(CH2)n- C - 15NH2 O
MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM
This is animated
XPA1-98
XPA RPA32C

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 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

29. 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!!!

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

31. 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.

32. 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

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

34. Flat Hydrophobic Contact Region
UDG
Peptide
RPA32C
Protein
Surface

35. Electrostatic Complimentarity
RPA32C
UDG
Peptide
Protein
Surface

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