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Open Dataset XS1A4OI7

Conformational assemblies of PARP1-DNA-HPF1-Timeless-EB47

Abstract

PARP1 detects DNA strand breaks and this leads to allosteric activation and synthesis of poly(ADP-ribose) modifications. HPF1 engages activated PARP1 to control target modification. Understanding of the mechanism of DNA break detection and catalytic activation is incomplete, due largely to limited structural information for full-length PARP1. Single-particle cryo-EM provided views of the full complement of PARP1 domains engaging a DNA single-strand break in the presence of HPF1 and a fragment of binding partner Timeless. However, it remained unclear to what extent the observed conformations existed in solution and in what proportions. This SEC-SAXS-MALS experiment confirmed an equimolar assembly that is dynamic and flexible. The Rg and P(r) profiles did not match any one static conformation, but did match assemblies of conformations when flexible linkers and the HD-ART hinge region were no longer fixed. See Sverzhinsky et al 2026, Nature Communications with the DOI below.

Experimental description

Full-length PARP1, nicked dumbbell DNA, and the type I inhibitor EB47 were assembled at 40 µM with 80 µM HPF1 and Timeless fragment and injected onto a Superdex 200 column in buffer (20 mM Hepes pH 8, 150 mM NaCl, 0.1 mM TCEP, 1 mM EDTA, and 10 µM EB47). The elution was analyzed in parallel by MALS and SAXS. For the latter, samples were exposed to 1.24 Å X-rays for 2 s with a sample-to-detector distance of ~2.1 m.

File description

A ZIP file containing the unsubtracted SAXS frames and three PDB files of the three starting models used in BilboMD simulations.

Created

2023-12-19

Published

2026-03-26

Data collection technique

SEC-SAXS

Journal DOI

https://doi.org/10.1038/s41467-026-69375-3

Source

Advanced Light Source

Beamline

SIBYLS BL12.3.1

Wavelength

1.24 Å

Sample to Detector Distance

2.1 m

Submitting Author

A. Sverzhinsky

Université de Montréal

Canada

Collaborators

J. Del Mundo, S. Classen, M. Hammel

Project Leader

J. Pascal

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Complete Set of SAS Data Files

The complete SAS dataset is downloadable as a zip file.

XS1A4OI7.zip Download

Individual SAS Data Files (Total: 0)

These are individual SAS data files that may be raw or processed (merged, etc.). These may or not be included in a zip file containing a larger dataset.

Supplemental Data and Supporting Materials (Total: 3)

These data and materials may include X-ray crystal structure coordinates, multi-angle light scattering data, .etc. It may also include additional details or methods pertaining to the SAXS experiments, or the researcher's interpretations of the results.

PARP1-dNick10_10-HPF1-Tmls_hybrid_compact.pdb Download
PARP1-dNick10_10-HPF1-Tmls_hybrid_extended.pdb Download
PARP1-dNick10_10-HPF1-Tmls_hybrid_predicted.pdb Download

Open SAXS data analysis sites in a new tab using these links

SAXS Similarity SAXS FrameSlice

Samples:

Macromolecule 1: HPF1

Sample Full Name: Histone PARylation factor 1
Sample Type: Protein
Source Organism: Homo sapiens
Source Organism NCBI Taxonomy ID: 9606
Expression System: Escherichia coli
Expression NCBI Taxonomy ID: 562
Uniprot ID: Q9NWY4
Sequence or Chemical Formula:
SMVGGGGKRRPGGEGPQCEKTTDVKKSKFCEADVSSDLRKEVENHYKLSLPEDFYHFWKFCEELDPEKPSDSLSASLGLQLVGPYDILAGKHKTKKKSTGLNFNLHWRFYYDPPEFQTIIIGDNKTQYHMGYFRDSPDEFPVYVGINEAKKNCIIVPNGDNVFAAVKLFLTKKLREITDKKKINLLKNIDEKLTEAARELGYSLEQRTVKMKQRDKKVVTKTFHGAGLVVPVDKNDVGYRELPETDADLKRICKTIVEAASDEERLKAFAPIQEMMTFVQFANDECDYGMGLELGMDLFCYGSHYFHKVAGQLLPLAYNLLKRNLFAEIIEEHLANRSQENIDQLAA

Macromolecule 2: PARP1

Sample Full Name: Poly [ADP-ribose] polymerase 1
Sample Type: Protein
Source Organism: Homo sapiens
Source Organism NCBI Taxonomy ID: 9606
Expression System: Escherichia coli
Expression NCBI Taxonomy ID: 562
Uniprot ID: P09874
Sequence or Chemical Formula:
MGSSHHHHHHSSGLVPRGSHMAESSDKLYRVEYAKSGRASCKKCSESIPKDSLRMAIMVQSPMFDGKVPHWYHFSCFWKVGHSIRHPDVEVDGFSELRWDDQQKVKKTAEAGGVTGKGQDGIGSKAEKTLGDFAAEYAKSNRSTCKGCMEKIEKGQVRLSKKMVDPEKPQLGMIDRWYHPGCFVKNREELGFRPEYSASQLKGFSLLATEDKEALKKQLPGVKSEGKRKGDEVDGVDEVAKKKSKKEKDKDSKLEKALKAQNDLIWNIKDELKKVCSTNDLKELLIFNKQQVPSGESAILDRVADGMVFGALLPCEECSGQLVFKSDAYYCTGDVTAWTKCMVKTQTPNRKEWVTPKEFREISYLKKLKVKKQDRIFPPETSASVAATPPPSTASAPAAVNSSASADKPLSNMKILTLGKLSRNKDEVKAMIEKLGGKLTGTANKASLCISTKKEVEKMNKKMEEVKEANIRVVSEDFLQDVSASTKSLQELFLAHILSPWGAEVKAEPVEVVAPRGKSGAALSKKSKGQVKEEGINKSEKRMKLTLKGGAAVDPDSGLEHSAHVLEKGGKVFSATLGLVDIVKGTNSYYKLQLLEDDKENRYWIFRSWGRVGTVIGSNKLEQMPSKEDAIEHFMKLYEEKTGNAWHSKNFTKYPKKFYPLEIDYGQDEEAVKKLTVNPGTKSKLPKPVQDLIKMIFDVESMKKAMVEYEIDLQKMPLGKLSKRQIQAAYSILSEVQQAVSQGSSDSQILDLSNRFYTLIPHDFGMKKPPLLNNADSVQAKVEMLDNLLDIEVAYSLLRGGSDDSSKDPIDVNYEKLKTDIKVVDRDSEEAEIIRKYVKNTHATTHNAYDLEVIDIFKIEREGECQRYKPFKQLHNRRLLWHGSRTTNFAGILSQGLRIAPPEAPVTGYMFGKGIYFADMVSKSANYCHTSQGDPIGLILLGEVALGNMYELKHASHISKLPKGKHSVKGLGKTTPDPSANISLDGVDVPLGTGISSGVNDTSLLYNEYIVYDIAQVNLKYLLKLKFNFKTSLW

Macromolecule 3: hTIM

Sample Full Name: Protein timeless homolog
Sample Type: Protein
Source Organism: Homo sapiens
Source Organism NCBI Taxonomy ID: 9606
Expression System: Escherichia coli
Expression NCBI Taxonomy ID: 562
Uniprot ID: Q9UNS1
Sequence or Chemical Formula:
SLVLSNENLGQSLHQEGFSIPLLWLQNCLIRAADDREEDGCSQAVPLVPLTEENEEAMENEQFQQLLRKLGVRPPASGQETFWRIPAKLSPTQLRRAAASLSQPE

Macromolecule 4: dNick10_10

Sample Full Name: 48-mer DNA
Sample Type: DNA
Source Organism:
Source Organism NCBI Taxonomy ID:
Expression System:
Expression NCBI Taxonomy ID:
Uniprot ID's:
Sequence or Chemical Formula:
GCTGAGCTTCTGGTGAAGCTCAGCTCGCGGCAGCTGGTGCTGCCGCGA

Macromolecule 5: EB-47

Sample Full Name: 2-[4-[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]carbonylpiperazin-1-yl]-N-(1-oxidanylidene-2,3-dihydroisoindol-4-yl)ethanamide
Sample Type: Other
Source Organism:
Source Organism NCBI Taxonomy ID:
Expression System:
Expression NCBI Taxonomy ID:
Uniprot ID's:
Sequence or Chemical Formula:
C24H27N9O6