Samuel H. Wilson · 2007. 6. 21. · 1980s: cDNA cloning; expression in E. coli; FPLC at room...
Transcript of Samuel H. Wilson · 2007. 6. 21. · 1980s: cDNA cloning; expression in E. coli; FPLC at room...
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“History of DNA Repair”
Samuel H. WilsonLaboratory of Structural Biology
NIEHS, NIH, DHHS
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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A reverse transcriptase with unique enzymatic specificity compared with other RTs, Pol I & the known cellular
polymerase: Template use; KClresistance; metal ion specificity.
Hence, DNA polymerases have unique fingerprints with regard to catalytic
specificity.
(endogeneous retroviral particle in some mouse tumors)
Looking for cellular RT…
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Strong DNA polymerase activity…
incubationtime
protein dNTP
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Catalytic specificity of DNA polymerases
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Pol alpha
Pol betaPol delta
Pol gamma
Looking for a Cellular RT…
Fractionationof crude extract:Assays specific forRT and Pols alpha &beta.
1974
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Pol ß
EndogenousMuLV-likeRetroviral
RT
Pol γ
Pol α1
ActivatedDNA as T-P
Poly (rA)-oligo (dT)as T-P
Pol α2
3 DEAE-cellulosefractions
3 Phosphocellulose columns
F-T Lo-salt Hi-salt
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Pol γ
1975
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1979
purification and kinetics
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Yield in Fraction VI: ~5 µg/200 g tissue or batch (!)
Pol ßPol ß
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Ordered sequential mechanism
DNA addsfirst…
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Two Pol α species: Native Mr inrange of 190,000, both with
subunits in the range of50,000.
Questions: Is Pol ß a proteolyticfragment of Pol α ?
What is the Mr of the catalytic subunit of Pol α ?
1979
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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1980
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39-kDa Pol ßtryptic peptidesin black in eachpanel
Conclusion: Pol ß and Pol α are distinct at the primary structure level
Peptide mapping from SDS-PAGE gel slices
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Calf thymus/peptide mapping
1982
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Conclusion: Predicted Mr of catalytic subunit = ~180,000
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SDS-PAGE, as usual, but with DNA in the gel; then, renature in aDNA polymerase reaction mixture containing 32P-labeled dNTPs.
Wash and conduct autoradiography.
1983
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Activity gel (SDS-PAGE) analysis with purified enzymes (positive controls)
Pol ß
Klenow
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Activity gel analysis of calf thymus crude extract
Pol α (?)
Pol ß
?
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IP with monoclonal ab.using extract from35S-labeled cells.
Renature ~190,000 peptidefrom SDS-PAGE gel slice.
1984
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Brief exposure
Longer exposure
Monoclonal antibody IP of extract from 35S-labeled cells
Pol α
Pol ß
?
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Activity
Pol alpha activity found in the ~190,000 peptide
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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1986
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Example of phage λ plaque purification
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1988
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*
Induction@ 42°C1h 2h 3h
Example with rat enzyme
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M= 10 µg each
31-kDa domain
Starting sampleF-T“Polishing of purified Pol ß:”
Routine FPLCchromatography
(1987)
Yield: ~100 mg per batch(1 liter culture) !
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DNA Polymerase ß(335 residues; 39 kDa)
N C33590 149 262
HhH HhH
Polymerase (31-kDa)
Lyase (8-kDa)
D NC
Domain
Function•dRP Lyase•5’-Phosphate
Recognition•DNA Binding
•DNA Binding •DNA Synthesis •dNTP Selection
Subdomain
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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Ternary Complex Structure
Structure of substrate complex: Enzyme; oligonucleotide DNA; and correct incoming dNTP
GG
ddCTP
GG
ddCTPCdd
Polymerase + DNA + ddCTP/Mg2+
Trapped intermediatewith ddCMP at the primer
terminus.
(collaboration with Joe Kraut, Huguette Pelletier & Mike Sawaya)
(Project started in 1987 with phone call from Joe Kraut)
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Science, 264:1891-1903, 1994
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Model for pre-catalytic complex (atoms in red postulated)
“Catalytic”
“Nucleotide”
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Characterizing BER DNA synthesis using crystallography
Gapped and nicked BER intermediates used as DNA substrate
+ ddCTP and MgCl2
+ Pol βGG3'
GGCdd
ddCTP
Ternary complex with 1 nt. gap
2-nt gap
(Sawaya et al., Biochemistry, 36:11205-11215, 1997)
(Also obtained binary complexes with nicked DNA)
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Local Conformational Change Upon Formation of aTernary Complex with a Watson-Crick Base Pair
Ternary Complex: CLOSEDTernary Complex: CLOSED
Binary DNA Complex: OPEN
α-helix N
“Induced fit” hypothesisfor DNA synthesis
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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Extract mediated Uracil-DNABER and use of a neutralizingpolyclonal antibodies to Pol ß
1995
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32P-labeled dCMP incorporation into a 51-bp oligonucleotide product molecule
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Repair Product
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Nature, 379:183-186, 1996
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Repair Product
Intermediate
Complementation in Pol ß -/- MEF line with cDNA expression vector
Uracil-DNA BERusing 51-bp oligoas substrate: Uat position 22.
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
![Page 46: Samuel H. Wilson · 2007. 6. 21. · 1980s: cDNA cloning; expression in E. coli; FPLC at room temperature 1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-](https://reader036.fdocuments.net/reader036/viewer/2022062402/5fc1596f25261204af041bf4/html5/thumbnails/46.jpg)
JBC, 271:12213-12220, 1996
Role of Arg283in catalysis andfidelity
R283A is deficient in catalyticefficiency and in fidelity.
Explanation through crystal structure analysis.
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“ Tight fit for new base pair”
Y271Y271
Template Base P P -- P P -- PPIncomingIncomingBaseBase
R283R283 K280K280 N279N279 D276D276
Primer BasePrimer Base
α-Helix N
R283Aabolishes
key templatestrand contacts
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
![Page 49: Samuel H. Wilson · 2007. 6. 21. · 1980s: cDNA cloning; expression in E. coli; FPLC at room temperature 1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-](https://reader036.fdocuments.net/reader036/viewer/2022062402/5fc1596f25261204af041bf4/html5/thumbnails/49.jpg)
Summary of Pol ß Interactions with BER Factors(most were first identified by immunoprecipitation)
DNA Lig.III/XRCC1
(Lindahl lab)NEIL1/NEIL2
(Mitra/Wilson labs)PARP-1
(de Murcia lab)
P53(Prives lab)
WRN(Bohr/Wilson labs)
APE(Demple lab) PCNA
(Wilson/Tomkinsonlabs)
DNA Lig. I(Wilson lab)
Pol β FEN-1(Bohr lab)
AAG(Samson/Wilson labs)
HMGB1(Wilson lab)
9-1-1(Hubscher lab)
APC(Narayan lab)
HS70(Bases lab)
YB-1(Hazra/Wilson labs)
And others ?
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2'-deoxyuridine-5'-[(α,β)-imido]triphoshphate(dUMPNPP)
Ternary complex structure with non-hydrolyzable dUTPanalog, dUMPNPP, as incoming nucleotide opposite template
base dA (complex has 2 Mg ions and 3’OH of the primer).
Approach
N
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Significance of Structure with Primer O3’ intact: Both Metals in Position to Stabilize Penta-coordinated Transition State, Enabling Ouantum Mechanical Calculations of Transition State Development
Significance of Structure with Primer O3’ intact: Both Metals in Position to Stabilize Penta-coordinated Transition State, Enabling Ouantum Mechanical Calculations of Transition State Development
PrimerTerminus
In position for“in-line attack” by O3’
Batra et al., Structure, 14:757-766, 2006
Two Metals
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Pol ß+
X5'
X = G
Binary ComplexCrystals
Ternary ComplexCrystals
StructureDetermination
dAMPCPPdAMPCPP (non(non--hydrolyzablehydrolyzable) & Mn) & Mn2+2+
Ternary Complex Structure with Mismatched Incoming Nucleotide
Ternary Complex Structure with Mismatched Incoming Nucleotide
Batra et al. (in preparation)
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dA—dUMPNPP (Right) dGdG——dAMPCPPdAMPCPP (Wrong)(Wrong)
3.9 Å
3.4 Å
2.8 Å
O3’
O3’
Mn2+
orMg2+
Pα
Polymerase Active Site
Batra et al. (in preparation)
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
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Structure of the Photoreactive dCTP Analogue (FAB-dCTP) and Oligonucleotide DNA
(Exo-N-[ß-(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5’-triphosphate)
X = THF
Olga Lavrik &associates
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Photoaffinity Labeling of Proteins in Extracts(identification of HMGB1 as “Unknown Band I)
X = THF
1: Extract without dC*TP (control)2: Extract from Pol ß +/+ MEF 3: Bovine testis nuclear extract (HMGB1 negative)
Extract
Sequencing target (HMGB1)
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HMGB1 identified using MALDI/MS and MALDI/MS/MSBand #
1
2
Protein
Actin
HMGB1
Score
226
102
12
WholeLysate
MrMarkers
StreptavidinEluate
32P-label --
Mr
41609
24892
Sequence Coverage: 27%Red: Matched peptides shown
Underlined: MS/MS
1 MGKGDPKKPR GKMSSYAFFV QTCREEHKKK31 HPDASVNFSE FSKKCSERWK TMSAKEKGKF61 EDMAKADKAR YEREMKTYIP PKGETKKKFK91 DPNAPKRPPS AFFLFCSEYR PKIKGEHPGL121 SIGDVAKKLG EMWNNTAADD KHPYEKKAAK151 LKEKYEKDIA AYRAKGKPDA AKKGVVKAEK181 SKKKKEEEED EEDEEDEEEE EDEEDEEEEE211 DDDDE
-50 K
-40 K
(by Leesa Deterding & Ken Tomer)
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OGG1 HMGB1NTH1
Accumulation of GFP-tagged human proteins following laser-induced DNA damage
(by Akira Yasui and associates)
Prasad et al. (In Press)
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OGG1 HMGB1 KU70NTH1 RAD52
Accumulation of GFP-tagged human proteins at DNA damage sitesafter laser irradiation in the presence of a photo-sensitizer
Photo-sensitizer: 8-MOP (methoxypsoralen)10 scans in the presence of a photo-sensitizer
(by Akira Yasui and associates)
Prasad et al. (In Press)
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Transforming technologies along the way: A story about Pol ß & BER
1970s: Column chromat. (DEAE & PC); peptide mapping; activity gel assay; IPs and immunoaffinity purifications
1980s: cDNA cloning; expression in E. coli; FPLC at room temperature
1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-specific DNA; genetically modified mouse & cell models; structure-function analysis (structure guided amino acid alterations & kinetics)
2000—: IPs & blotting; crystallography with non-hydrolyzable dNTPs; sub-nuclear imaging; affinity capture & mass spectrometery
Case in point: A novel co-factor in mammalian BER (HMGB1)
i) Identification by interaction with BER intermediate and MSii) Recruitment at sites of DNA damage in a living cell
![Page 61: Samuel H. Wilson · 2007. 6. 21. · 1980s: cDNA cloning; expression in E. coli; FPLC at room temperature 1990s: Crystallography-ternary complex with dideoxy primer; lesion/site-](https://reader036.fdocuments.net/reader036/viewer/2022062402/5fc1596f25261204af041bf4/html5/thumbnails/61.jpg)
ACKNOWLEDGEMENTS
• Current associates• Former associates• Mentors• Friends & Colleagues• Competitors• Funding (NIH & foundations)
• Current collaborators• Former collaborators• Service cores• Commercial sources• Editors & journals
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DNA Repair Publications
0
1000
2000
3000
4000
5000
6000
7000
8000
1962
1966
1970
1974
1978
1982
1986
1990
1994
1998
2002
2006
Year
SCOPUS searched the following terms:TITLE-ABS-KEY (dna repair OR dna damage OR dna adducts
OR dna lesions OR nucleotide excision repair OR base excision repair OR mismatch repair). Total = 105,433
(Ben Van Houten)