slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies) PowerPoint Presentation
Download Presentation
Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies)

Loading in 2 Seconds...

play fullscreen
1 / 37

Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies) - PowerPoint PPT Presentation


  • 180 Views
  • Uploaded on

March 6-11, 2003. Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies). Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@ vanderbilt.edu http://structbio.vanderbilt.edu/chazin.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Biochemistry 305 Structural Mechanisms of the DNA Replication, Recombination and Repair (DNA Processing Assemblies)' - royce


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

March 6-11, 2003

Biochemistry 305Structural Mechanisms of the DNA Replication, Recombination and Repair(DNA Processing Assemblies)

Walter Chazin

5140 BIOSCI/MRBIII

E-mail: Walter.Chazin@ vanderbilt.edu

http://structbio.vanderbilt.edu/chazin

processing of dna is performed by multi protein assemblies
Processing of DNA is Performed by Multi-protein Assemblies

XPC

TFIIH

XPA

T Ag

XPF

Pol/ Prim

SSB

XPG

SSB

Replication

Repair

51

51

51

51

51

51

RAD52

51

SSB

How do

they work?

Recombination

dna processing involves common steps
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
dna processing is dynamic

T Ag

T Ag

SSB

SSB

SSB

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

SSB

DNA Processing is Dynamic

T Ag

SV40 Replication

  • Structural snapshots at different time points (X-ray, EM)
  • Need to characterize dynamic progression (NMR)
mechanism of dna processing preformed assemblies
Mechanism of DNA ProcessingPreformed Assemblies?

1

XPC

3

2

TFIIH

5

XPA

XPF

XPG

4

SSB

3,4,5….

  • How does progression occur?
  • Reorganization of a static complex?
mechanism of dna processing dynamic assembly
Mechanism of DNA ProcessingDynamic Assembly?

1

XPC

3

2

TFIIH

5

XPA

XPF

XPG

4

SSB

3,4,5….

Progression through the multiple steps by dynamic asembly/disassembly of sub-complexes

multiple proteins multiple steps

1

XPC

3

2

TFIIH

5

XPA

XPF

XPG

4

Multiple Proteins/Multiple Steps

1. Recognize damage

2. Unwind duplex

3. Locate lesion

4. Excise 5’

SSB

Nucleotide

Excision

Repair

5. Excise 3’

3,4,5….

What enables progress through the multiple steps?

structural approach to mechanism integrated use of nmr computation and crystallography
Structural Approach To MechanismIntegrated use of NMR, computation and crystallography

NMR

Computation

Crystallography

Biophysical Analysis

Expression/Mutations

RPA-A

RPA-B

Fluorescence Intensity

RPA-AB

Ratio of T-ag/RPA

where to start select the common element ssb
Where to Start?Select the Common Element (SSB)

XPC

TFIIH

XPA

XPF

T Ag

Pol/ Prim

SSB

XPG

SSB

Replication

Repair

51

51

51

51

51

51

RAD52

51

SSB

Recombination

the eukaryotic ssb is replication protein a rpa
The Eukaryotic SSB isReplication Protein A (RPA)

XPC

TFIIH

XPA

XPF

T Ag

Pol/ Prim

RPA

XPG

RPA

Replication

Repair

51

51

51

51

51

51

RAD52

51

RPA

Recombination

replication protein a rpa structure
Replication Protein A (RPA) Structure

OB-Fold

Winged HLH

Binds ssDNA

Binds proteins

  • Domains defined by limited proteolysis
  • Biochemical functions of domains assigned
slide12

3D Structures of RPA Domains

X-Ray

14/32D/70C

70AB

NMR

Zn

B

A

C

D

RPA70

RPA32

RPA14

NTD

14

CTD

32CTD

70NTD

quaternary structure?

refresher on nmr
Refresher on NMR

15N- 1H HSQC Spectrum

R

R

15N - Ca- CO - - -15N - Ca

H

H

15N Frequency

1H Frequency

  • One peak for each residue
  • Each peak can be assigned to a single site in the protein
nmr analysis of quaternary structure

C

RPA32

P

RPA14

NMR Analysis Of Quaternary Structure

15N RPA32/14

40

173

  • Flexibly linked domains: RPA32C is structurally independent of the core of RPA32/14
slide15

P

RPA: Coordinated Activity of Modules

14/32D/70C

70AB

Zn

B

A

C

D

RPA70

RPA32

RPA14

NTD

14

CTD

70NTD

32CTD

understanding structural mechanisms of dna processing assemblies
Understanding Structural Mechanisms Of DNA Processing Assemblies

1. Modularity: multiple domains, separate functions

interaction of rpa with damage recognition protein xpa
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

method to identify sites of interaction matrix of rpa affinity chromatography columns

Proteolysis

Target

Immobilized

RPA

Wash

Elute

Method to Identify Sites of InteractionMatrix of RPA Affinity Chromatography Columns

RPA Library

RPA14/32

RPA14/32DC

RPA14/32/70DN

RPA70DC

RPA32C

RPA70N

RPA70AB

Mass Spec

results using rpa14 32 as bait
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

rpa32c binds xpa n terminal region
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

interaction of rpa with xpa

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

understanding structural mechanisms of dna processing assemblies1
Understanding Structural Mechanisms of DNA Processing Assemblies
  • Modularity: multiple domains, separate functions

2. Multiple contact points: XPA

3d structure of rpa32c the start not the end

C

N

Winged Helix-Loop-Helix

3D Structure of RPA32CThe Start - Not The End!!
  • What is the molecular basis for interaction?
quantifying rpa32c xpan interaction nmr titration 15 n rpa32c xpan
Quantifying RPA32C-XPAN InteractionNMR Titration: 15N-RPA32C + XPAN

RPA32C

RPA32C + XPA 1-98

R

R

15N - Ca- CO - - -15N - Ca

H

H

  • Determine affinity (20 mM )
  • Map the binding site
    • Only 19 residues affected  discrete binding site

Mer et al., Cell 2000

map binding site on the structure

C

N

Map Binding Site On The Structure

RPA32C

Discrete Binding Site

  • Small surface on XPA
  • Could be a helical element from XPA
titration of 15 n xpa 1 98 rpa32c reveals side chains in binding site
Titration of 15N-XPA1-98 + RPA32C Reveals Side Chains in Binding Site

O

(CH2)n- C - 15NH2

-15NH- Ca- CO -

XPA1-98

XPA1-98 + RPA32C

MAAADGALPEAAALEQPAELPASVRASIERKRQRALMLRQARLAARPYSATAAAATGGMANVKAAPKIIDTGGGFILEEEEEEEQKIGKVVHQPGPVM

peptide induces same shifts in rpa32c as xpa n terminal domain
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

predict binding sites in other dna repair proteins
Predict Binding Sites in Other DNA Repair Proteins

ERKRQRALMLRQARLAAR

RIQRNKAAALLRLAAR

RKLRQKQLQQQFRERMEK

NER

XPA29-46

UDG79-88

RAD257-274

BER

RR

Patterns But Not Homology!!!

flat hydrophobic contact region

UDG

Peptide

RPA32C

Protein

Surface

Flat Hydrophobic Contact Region
electrostatic complimentarity

RPA32C

UDG

Peptide

Protein

Surface

Electrostatic Complimentarity
slide36

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

understanding structural mechanisms of dna processing assemblies2
Understanding Structural Mechanisms Of DNA Processing Assemblies

1. Modularity: multiple domains, separate functions

2. Multiple contact points: XPA

3. Modest affinity: micromolar contact points