Understanding Structural Mechanisms Of DNA Processing Assemblies

Understanding Structural Mechanisms Understanding Structural Mechanisms Of DNA Processing AssembliesOf DNA Processing Assemblies

1. Modularity: multiple domains, separate functions

2. Multiple contact points: XPA

3. Modest affinity: micromolar contact points

C BZn

A

NTD

14CTD

D

70NTD

70AB14/32D/70C

32CTD

RPA: Coordinated Activity of ModulesRPA: Coordinated Activity of Modules

RPA70RPA70 RPA32RPA32RPA14RPA14

P

Proteolysis

XPAN - RPA32CXPAC - RPA70N

EluteWash

XPA

RPA LibraryRPA LibraryRPA14/32RPA14/32RPA14/32RPA14/32CCRPA14/32/70RPA14/32/70NNRPA70RPA70CCRPA32CRPA32CRPA70NRPA70NRPA70ABRPA70AB

ImmobilizedImmobilizedRPA RPA

Interaction of RPA with XPAInteraction of RPA with XPA

RPA Modulates Function inRPA Modulates Function inMultiple DNA Repair PathwaysMultiple DNA Repair Pathways

NER

BER

RR

RPA32RPA32

XPAXPA29-4629-46UDGUDG79-8879-88 RADRAD257-274257-274

A Common Mode of Interaction for A Common Mode of Interaction for Different DNA Repair ProteinsDifferent DNA Repair Proteins

RPA Drives Assembly and Commitment RPA Drives Assembly and Commitment to a Specific Pathway of Repairto a Specific Pathway of Repair

NER

BER

RR

RPA32RPA32

RPA32C- Built for Dynamic Binding RPA32C- Built for Dynamic Binding Simple Motif / Modest AffinitySimple Motif / Modest Affinity

Central, flat hydrophobic surface

Electrostatic complimentarity at either end of the binding region

Mer et al., Cell 2000




3. Modest affinity: micromolar contact points

4. Multiple interactions: multiple proteins, DNA

Using Concepts To Build ModelsUsing Concepts To Build ModelsUDG: A Modular Damage Recognition ProteinUDG: A Modular Damage Recognition Protein

• Multiple contacts with relatively modest affinity • Modularity allows integration of multiple functions

UDG

Continuing to Build View of NERContinuing to Build View of NER RPA Interactions with XPG and XPF/ERCC1RPA Interactions with XPG and XPF/ERCC1

TFIIH

2XPF

5XPC

1

XPA

3

XPG

4

RPA

3,4,5….3,4,5….

1. Recognize damage

3. Locate lesion3. Locate lesion

4. Excise 5’4. Excise 5’

5. Excise 3’5. Excise 3’

2. Unwind duplex

• Cannot all be independent sites on RPA!• Direct competition for sites, other mechanisms?




3. Modest affinity: micromolar per contact point


Forward progression: structural transitions

Binding of ssDNABinding of ssDNA

RPA Binds DNA Non-SpecificallyRPA Binds DNA Non-Specifically

• Needs to bind ssDNA regardless of sequence!!

Structure Shows Base-Specific Contacts!Structure Shows Base-Specific Contacts! OB-Fold Base-specific contacts

• Needs to bind all ssDNA sequences!!! (X-ray and NMR)

NMR Reveals Solution Same as CrystalNMR Reveals Solution Same as Crystal

• NMR can be used to study ssDNA binding properties

NMR Assessment of 3 ssDNA OligomersNMR Assessment of 3 ssDNA Oligomers

• RPA70AB binds all ssDNA in the same manner

ssDNA Binding Site Is Highly DynamicssDNA Binding Site Is Highly Dynamic

0.4

0.6

0.8

1.0

0.4

0.6

0.8

1.0

183 203 223 243 263 283

S2

S2

• Binding of ssDNA quenches motions

Mechanism for Non-Specific BindingMechanism for Non-Specific Binding

Hypothesis

The intrinsically flexible binding site is able to

remodel in response to the properties of

different DNA bases

Binding of ProteinsBinding of Proteins

What Structural Mechanisms Allow What Structural Mechanisms Allow Progression Of SV40 Replication?Progression Of SV40 Replication?

RPA

RPA

RPA

T AgT Ag

T AgT AgT Ag T Ag

Pol/ Prim

RPA

Pol/

PrimT AgT AgT Ag

• Analyze interactions of RPA with SV40 Large T-antigen

The SV40 T-ag Origin Recognition The SV40 T-ag Origin Recognition Domain Binds to RPA70ABDomain Binds to RPA70AB

RPA-A RPA-B RPA-AB

Con

trol

+Try

psin

Con

trol

+Try

psin

Con

trol

+Try

psin

+T

-ag

+Try

psin

+Try

psin

+T

-ag

+Try

psin

+T

-ag

+Try

psin

+T

-ag

+Try

psin

+T

-ag

+Try

psin

+T

-ag

Affinity of RPA70AB For SV40 T-ag Affinity of RPA70AB For SV40 T-ag Origin Recognition Domain Is ModestOrigin Recognition Domain Is Modest

No

rmal

ized

RP

A F

luo

resc

ence

Molar Ratio of T-ag

NMR Analysis of Structure and BindingNMR Analysis of Structure and Binding

• Not all residues affected, ~100 M affinity

1515N T-agN T-ag131-259131-259

15N

1H

15N RPA70AB

Mapping Binding Sites on StructuresMapping Binding Sites on Structures

Bochkarev et al., 1997 Luo et al., 1996

T-agT-ag131-259131-259 RPA70ABRPA70AB

• Discrete binding sites, modest affinity• T-ag binds remote from the ssDNA binding site

Is T-ag an Allosteric Effector of RPA?Is T-ag an Allosteric Effector of RPA?

Bochkarev et al., 1997 Luo et al., 1996

T-agT-ag131-259131-259 RPA70ABRPA70AB

Binding of T-ag Alters Affinity for ssDNA Binding of T-ag Alters Affinity for ssDNA

No

rmal

ized

RP

A F

luo

resc

ence

Molar Ratio of ssDNA

d-CTTCACTTCA + T-ag131-259

d-CTTCACTTCA

• Pre-loading T-ag increases RPA’s affinity for ssDNA

• Converse: releasing T-ag lowers affinity for ssDNA

Structural Stabilization From BindingStructural Stabilization From Binding

• Tighter binding gives rise to equal or BETTER!! spectra in >40 kDa ternary complex

15N

RPA70AB/ssDNA + T-ag RPA70AB/T-ag + ssDNA

What is the mechanism for allostery?What is the mechanism for allostery?

Mechanism: Independent DomainsMechanism: Independent Domains

• A and B domains behave as independent modules (in the absence of binding partners)

RPA70AB RPA70ARPA70A + + RPA70BRPA70B

Mechanism: ssDNA Binding RequiresMechanism: ssDNA Binding RequiresAlignment of A and B DomainsAlignment of A and B Domains

Different interdomain angles

ssDNAssDNA bound with 5’-3’ polarity

domains aligned

Pre-loading T-ag on RPA70AB pre-aligns the A and B domains

Entropic Contribution To AllosteryEntropic Contribution To Allostery

Bochkarev et al., 1997

RPA70ABRPA70AB

Lower penalty for loss of entropy = higher DNA affinity

Model for Dynamic Progression Model for Dynamic Progression From Unwinding to PrimingFrom Unwinding to Priming

T Ag

T Ag T Ag

T AgT AgT Ag T AgT Ag T Ag

RPA

• Pol-prim out-competes T-ag for RPA, which causes release of ssDNA and “hand-off” to DNA primase step

RPA

RPA

T AgT Ag

T AgT AgT Ag T Ag

Pol/ Prim

RPA

RPA

T AgT Ag

T AgT AgT Ag T Ag

Pol/

Prim

Next Step: Extend Analysis to PrimaseNext Step: Extend Analysis to Primase

RPA

RPA

RPA

T AgT Ag

T AgT AgT Ag T Ag

Pol/ Prim

RPA

Pol/

PrimT AgT AgT Ag

• Identify interaction modules, characterize binding

The Essential Recombination Factor The Essential Recombination Factor Rad51N Also Interacts With RPA70ABRad51N Also Interacts With RPA70AB

• Are the structural mechanisms the same as for T-ag?

RAD52

RPA51

5151 51 51 51 51

Pre-loading Rad51N on RPA70AB Pre-loading Rad51N on RPA70AB Affects the Binding Affinity for ssDNAAffects the Binding Affinity for ssDNA

Molar Ratio of ssDNA

d-CTTCACTTCA + Rad51N

No

rmal

ized

RP

A F

luo

resc

ence

But Rad51 Binds Differently to RPA70AB!!But Rad51 Binds Differently to RPA70AB!!

Bochkarev et al., 1997

Aihara et al., 1999

Mechanism must be differentAllostery versus direct competition for sites?

15N RPA70AB

RPA70AB Rad511-93

Structural Mechanisms of DNA Structural Mechanisms of DNA Processing Assemblies: Key ConceptsProcessing Assemblies: Key Concepts


2. Multiple contact points: XPA, T-ag, DNA primase

3. Modest affinity: micromolar per contact point


5. Structural transitions: direct competition between sites; allosteric coupling

Model for Progression ofModel for Progression ofDNA Processing AssembliesDNA 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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Understanding Structural Mechanisms Of DNA Processing Assemblies