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Structural and Biochemical Studies to Assess Structural and Biochemical Studies to Assess Protein Interactions and (re)classify VUSs Aishwarya Prakash Assistant Professor of Oncologic Sciences Mitchell Cancer Institute DNA Repair Video Conference Feb 19th, 2019 Outline • PART I: Specialized functions of the NEIL1 DNA Glycosylase – BER & NEIL1 – The interaction between NEIL1 and mitochondrial SSB, in vitro – Concluding remarks • PART II: (Re)Classification of Variants of Uncertain Significance in Lynch syndrome patients – Identification of Variants of Uncertain Significance – Functional Characterization – Concluding remarks DNA Damage and Repair Pathways Reactive Oxygen Species ROS X-rays Ionizing Radiation Replication Errors Anti-tumor Drugs UV Light T T G T Mismatch Repair Excision Recombinational Nucleotide Excision (MMR) Repair (BER, Repair (HR, NHEJ) Repair (NER) SSBR) Adapted from Tubbs, Cell 2017; https://diethylstilbestrol.co.uk/dna-repair-system/ PART I: Base excision repair & NEIL1 5’ 3’ 3’ 5’ Monofunctional glycosylases Bifunctional glycosylases MYH NEIL1 MPG TDG OGG1 UNG MBD4 NEIL2 NTH1 NEIL3 SMUG1 5’ 3’ β-elimination βδ-elimination 3’ 5’ 5’ P 3’ 5’ P P 3’ APE1 3’ 5’ 3’ 5’ OH APE1 5’ dRP 3’ PNK 3’ 5’ Polβ Polβ OH P Polδ 5’ 3’ 5’ 3’ Polε 3’ 5’ 3’ 5’ Polβ Polβ 5’ 3’ 3’ 5’ 5’ P 3’ FEN1 3’ 5’ 5’ P 3’ Lig III / XRCC1 3’ 5’ 5’ 3’ Lig I 3’ 5’ Short-Patch BER 5’ 3’ 3’ 5’ Long-Patch BER Adapted from Hegde ML, Cell Research, 2008 and Duclos et al., 2011 Structural features of the Fpg/Nei DNA glycosylase family H2TH DNA binding motif EcoNeI 263 aa EcoFpg 269 aa Zincless finger NEIL1 390 aa Zinc finger NEIL2 332 aa Ran-GTP Zinc finger NEIL3 605 aa H2TH Blue – NEIL1 Pink - EcoFpg Zinc finger PDB: 1R2Y 5ITX C-terminal Domain N-terminal Domain Fromme, JC, Verdine, GL. (2003) J Biol Chem 278 51543-51548 Zhu, C. et. al. (2016) Proc.Natl.Acad.Sci.USA 113: 7792-7797 The C-terminal tail of NEIL1 is involved with protein interactions 2 334 2 NEIL1-△56 290 390 NEIL1 290 Polδ/Lig I 390 312 349 FEN-1/RPA/WRN/RF-C 290 PCNA 349 C-terminal disordered region Prakash A. et al., NAR. 2017 Hegde M. et al., JMB. 2013 Sharma N. et al., DNA Repair. 2018 Doublié S. et al., PNAS. 2004 Doublié S. et al., PNAS. 2004 Adapted from Rangaswamy et al., Genes. 2017 Role of NEIL1 in mitochondrial DNA repair Question: Does NEIL1 interact with proteins associated with mtDNA replication? Nidhi Sharma Some proteins involved with mitochondrial DNA maintenance mtSSB, Homotetramer, PDB: 3ULL Polymerase Gamma, Heterotrimer, PDB: 5C51 Twinkle helicase, Nucleic Acids Res. 2015 Apr 30; 43(8): 4284–4295. Transcription factor A (TFAM), PDB: 3TMM Complement Component 1, q subcomponent binding protein (C1QBP), homotrimer, PDB: 3RPX Sharma N. et al. DNA Repair. 2018 Prakash Lab, unpublished. 2018 A combined approach to studying the interaction between NEIL1 and mtSSB Structural Interactions between NEIL1 and mtSSB in solution • Protein Painting & Far-western analysis • Size-exclusion chromatography (SEC), Multi-Angle Light Scattering (MALS), & Small Angle X-ray Scattering (SAXS) NEIL1-DNA, PDB: 5ITX mtSSB Tetramer, PDB: 3ULL Yang C. et. al., Nat. Struct. Biol. 1997 https://fineartamerica.com/featured/p Zhu, C. et. al. Proc.Natl.Acad.Sci. 2016 rotein-structure-epstudio-design.html Protein painting to determine interface contacts Coat surface Denature/ with molecular Dissociate paints Reduction/ Alkylation/ Trypsin Binding NEIL1/2 Digestion Partner Protease cleavage Set A: Peptides from Set B: Peptides from individual proteins protein complex Mass Spectrometry B A B – A = Interface contacts Adapted from Luchini A., et al. Nature Communications 5, Article number: 4413 (2014) Examples of molecular protein paints RBB, ANSA, Anthraquinone Naphthalene Derivative CR (Congo red), AO50 (Acid Orange 50), Acryl azo Aryl azo compound compound MV (Methyl violet), Triarylmethane DECI, compound Polymethine compound Adapted from Luchini A., et al. Nature Communications 5, Article number: 4413 (2014) Protein Painting with NEIL1, mtSSB, and the N-terminal NEIL1-mtSSB complexC-terminal Tetramer interface Dimer interface mtSSB Structure (PDB ID: 3ULL) Protein painting with the NEIL1-mtSSB complex Full-length NEIL1 Model: RaptorX NEIL1 residues 289 – 312 are required for an interaction with mtSSB 390 NEIL1-FL 349 NEIL1-Δ40 334 NEIL1-Δ56 312 NEIL1-Δ78 289 NEIL1-Δ100 289 390 GST-289-389 312 390 GST-312-389 289 349 GST-289-349 312 349 GST-312-349 GST 75 50 37 25 20 Coomassie stain Far Western Sharma N. et al. DNA Repair. 2018 NEIL1:mtSSB complex formation via SEC Ø Size exclusion chromatography (SEC) ) separates molecules based on molecular weight and shape or Stokes radius. mAU Ø Molecules with bigger Stokes radii elute first Absorbance ( Absorbance Volume (ml) ü Complex formation between NEIL1-mtSSB 24 26 28 30 32 34 36 38 40 Fraction (no.) in absence of DNA was confirmed as the ) molecule is eluted as single peak. mAU 26 27 28 29 30 31 32 33 50 ü We noted a smaller Stokes radius for the 37 NEIL1-mtSSB complex. 25 Absorbance ( 15 Volume (ml) 24 26 28 30 32 34 36 38 40 Fraction (no.) ü Absence of the NEIL1 C-terminal 100 Theoretical molecular Protein/complexresidues abolishes the interaction with Elution Volume (ml) Stokes radius (Å) mtSSB weight (kDa) mtSSB 60.78 14.29 39.2 NEIL1 44.75 13.73 42.2 mtSSB-NEIL1 105 14.26 39.4 Sharma et. al. DNA Repair, 2018 Complex formation of NEIL1 and mtSSB in the presence of DNA ) TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAGACCTGGACG mAU CATCTGXACCTGC Absorbance ( Absorbance Volume (ml) Theoretical molecular Protein/complex Elution Volume (ml) Stokes radius (Å) weight (kDa) mtSSB 60.78 14.29 39.2 NEIL1 44.75 13.73 42.2 mtSSB-NEIL1-DNA 128 12.22 50.3 ü Size exclusion chromatography indicated the formation of a larger ternary complex (NEIL1-DNA- mtSSB) in presence of DNA Sharma et. al. DNA Repair, 2018 Information obtained from SEC-MALS-SAXS ØAbsolute molar mass and stoichiometry (MALS) ØMaximum particle dimension (Dmax; SAXS) ØSize and shape of molecule – P(r) function (SEC & SAXS) ØFlexibility/disorder (Kratky plot; SAXS) ØEstimation of molecular weight (MALS and SAXS) ØOligomerization state and organization in solution (MALS & SAXS) ØLow resolution molecular envelopes (Blob-o-logy; SAXS) SEC-SAXS/SEC-MALS-SAXS setup Sample/Complex SEC Detector at large distance Scattering Curves UV detector 2θ < 5 degrees mtSSB NEIL1 mtSSB-DNA I 10 NEIL1-mtSSB NEIL1-DNA-mtSSB X-rays Buffer subtraction RelativeLog Momentum Transfer (Å) Multi-angle light scattering • ATSAS Package/Scatter • Data analysis- I0, Rg, Dmax , MW • Ab-initio shape modeling Differential refractive index (dRI) Srinivas Chakravarthy, PhD BioCAT, Argonne National Labs Shape Information from SAXS Analysis 2D Scattering Curves Distribution of inter-atomic distances, p(r) Svergun DI, Koch MHJ. Rep. Prog. Phys. 66 (2003) 1735–1782 SEC-SAXS analysis for multimeric complexes mtSSB Ab ini&o shape NEIL1 mtSSB-DNA reconstruc0on NEIL1-mtSSB NEIL1-DNA-mtSSB Relativep(r) Å 150 Distance r (Å) Pair-wise Distance Distribu0on GNOM analysis mtSSB NEIL1 mtSSB-DNA NEIL1-mtSSB NEIL1-DNA-mtSSB I 2 q Kratky Analysis Momentum Transfer (Å) Sharma et. al. DNA Repair, 2018 http://www.bioisis.net/tutorial/6 Summary of SAXS and MALS data for NEIL1, mtSSB, and complexes mtSSB NEIL1 mtSSB-DNA NEIL1-mtSSB NEIL1-DNA- (tetramer) complex complex mtSSB complex Structural parameters Rg (Å) from 27.52 37.15 28.18 28.12 46.86 P(r) Rg from 27.44±1.21 33.04±2.46 28.40±0.86 28.48±0.80 44.83±1.80 GuInIer Dmax (Å) 103.21 149.96 98.74 94.32 165.72 Molecular weight determination (kDa) TheoretIcal 60.78 44.72 78 105.5 123 MW(Vp) 61 46 79 60.3 146.6 MW(Vc) 59 36 71 55.4 134.3 MW (MALS) 59.8±1.67% 48.5±2.23% 69±1.46% 58±1.54% 129.6±1.54% Sharma et. al. DNA Repair, 2018 Ab initio shape reconstruction of NEIL1, mtSSB, & complexes NEIL1 NEIL1-mtSSB NEIL1-mtSSB-DNA Oblate Oblate X2 = 1.072 X2 = 1.311 X2 = 1.072 mtSSB Prolate Prolate X2 = 1.041 X2 = 1.311 X2 = 1.066 mtSSB-DNA Unknown Unknown X2 = 0.945 X2 = 1.312 X2 = 1.069 Sharma et. al. DNA Repair, 2018 Conclusions and Significance Ø NEIL1 interacts with mtSSB via its disordered C- terminal tail (protein painting, far western, SEC-MALS) Ø Absolute molar mass values obtained via MALS indicate that NEIL1 interacts with a monomer of mtSSB Ø The NEIL enzymes may have acquired specific functions during cellular processes such as replication and transcription Ø NEIL1-mediated disruption of replication proteins indicates a potential mechanistic switch between DNA replication and repair Outline • PART I: Specialized functions of the NEIL1 DNA Glycosylase – BER & NEIL1 – The interaction between NEIL1 and mitochondrial SSB, in vitro – Concluding remarks • PART II: (Re)Classification of Variants of Uncertain Significance in Lynch syndrome patients – Identification of Variants of Uncertain Significance – Functional Characterization – Concluding remarks 5’ 3’ 3’ 5’ 5’ 3’ Mismatch Repair (MMR) Overview 5’ 3’ 5’ MutSa MutLa MutSb MutLa Leading Strand Synthesis MSH6 PMS2 MSH3 PMS2 MSH2 MLH1 MSH2 MLH1 T G Mismatch recognition T 1 2 4 G MSH6T MSH2 G base-base Insertion – deletion mispair mismatch Incision MSH6T PMS2 Polymerase d/e MSH2G MLH1 PCNA Removal MSH6 ExoI MSH6T MSH2 MutSa MSH2G G PMS2 MLH1 MutLa Resynthesis and Ligation ExoI C ExoI (5’ to 3’) G RPA Brandon D’Arcy, Adapted from Hsieh P et al., 2017, and http://erielab.web.unc.edu/research/ Lynch syndrome and cancer Heterozygous mutations in 1 of the 4 main MMR genes (MSH2, MSH6, MLH1, and PMS2) and EPCAM causes Lynch syndrome (LS).
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