Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF.

Corbeski I, Guo X, Eckhardt BV, Fasci D, Wiegant W, Graewert MA, Vreeken K, Wienk H, Svergun DI, Heck AJR, van Attikum H, Boelens R, Sixma TK, Mattiroli F, van Ingen H, Sci Adv 8(30):eabo0517 (2022) Europe PMC

SASDJJ5 – Histone H2A:H2B:H3:H4 complex with aprataxin and polynucleotide kinase like factor (APLF) acidic domain

Aprataxin and PNK-like factor (acidic domain)
Histone H2A
Histone H2B
Histone H3
Histone H4
MWexperimental 120 kDa
MWexpected 122 kDa
VPorod 260 nm3
log I(s) 7.94×102 7.94×101 7.94×100 7.94×10-1
Aprataxin and PNK-like factor (acidic domain) Histone H2A Histone H2B Histone H3 Histone H4 small angle scattering data  s, nm-1
ln I(s)
Aprataxin and PNK-like factor (acidic domain) Histone H2A Histone H2B Histone H3 Histone H4 Guinier plot ln 7.94×102 Rg: 3.7 nm 0 (3.7 nm)-2 s2
(sRg)2I(s)/I(0)
Aprataxin and PNK-like factor (acidic domain) Histone H2A Histone H2B Histone H3 Histone H4 Kratky plot 1.104 0 3 sRg
p(r)
Aprataxin and PNK-like factor (acidic domain) Histone H2A Histone H2B Histone H3 Histone H4 pair distance distribution function Rg: 3.7 nm 0 Dmax: 10.8 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Aprataxin and PNK-like factor (acidic domain) Histone H2A Histone H2B Histone H3 Histone H4 DAMMIF model

Synchrotron SAXS data from solutions of the H2A:H2B:H3:H4 histone complex bound to the acidic domain of APLF in 25 mM NaPi, 300 mM NaCl, 3% v/v glycerol, 1 mM DTT, pH 7 were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.12 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). 40 successive 1 second frames were collected from a sample and buffer at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentration were merged with the highest concentration high angle data to yield the final composite scattering curve.

The data show the merged SAXS data obtained from batch and SEC-UV-SAXS measurements and therefore the concentration is not specified. The SEC parameters were: Column type: GE Superdex S200 Increase 10/300. Flow rate: 0.6 ml/min. Injection volume and load concentration: 40 µl at 14 mg/mL. Total run time: 35 min. NOTE: The complex consists of one histone complex (8 subunits) and two bound APLF molecules. However, the APLF molecules are not in contact with each other and are a separated on the histone complex. The molecular weight of the complex was validated using mass spectrometry. The molecular weight, from native mass-spec gives 122,926.1 +/- 9.6 Da, for expected mass of 122,839.8 Da based on the atomic composition.

Aprataxin and PNK-like factor (acidic domain) (APLF-AD)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   7.6 kDa
 
UniProt   Q8IW19 (449-511)
Sequence   FASTA
 
Histone H2A (His2A)
Mol. type   Protein
Organism   Drosophila melanogaster
Olig. state   Dimer
Mon. MW   13.2 kDa
 
UniProt   P84051 (2-124)
Sequence   FASTA
 
Histone H2B (His2B)
Mol. type   Protein
Organism   Drosophila melanogaster
Olig. state   Dimer
Mon. MW   13.7 kDa
 
UniProt   P02283 (2-123)
Sequence   FASTA
 
Histone H3 (His3)
Mol. type   Protein
Organism   Drosophila melanogaster
Olig. state   Dimer
Mon. MW   15.2 kDa
 
UniProt   P02299 (2-136)
Sequence   FASTA
 
Histone H4 (His4)
Mol. type   Protein
Organism   Drosophila melanogaster
Olig. state   Dimer
Mon. MW   11.4 kDa
 
UniProt   P84040 (2-103)
Sequence   FASTA