Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase.

Prakash A, Moharana K, Wallace SS, DoubliƩ S, Nucleic Acids Res 45(5):2897-2909 (2017) PubMed

SASDBA7 – Human NEI like DNA glycosylase 1 (NEIL1) bound to Proliferating Cell Nuclear Antigen (PCNA) and DNA

Endonuclease 8-like 1
dsDNA
Proliferating cell nuclear antigen
MWI(0) 130 kDa
MWexpected 77 kDa
VPorod 113 nm3
log I(s) 7.07×102 7.07×101 7.07×100 7.07×10-1
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen small angle scattering data  s, nm-1
ln I(s)
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen Guinier plot ln 7.08×102 Rg: 3.4 nm 0 (3.4 nm)-2 s2
(sRg)2I(s)/I(0)
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen Kratky plot 1.104 0 3 sRg
p(r)
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen pair distance distribution function Rg: 3.8 nm 0 Dmax: 16.4 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen DAMMIN model

log I(s)
 s, nm-1
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen DAMMIN model

log I(s)
 s, nm-1
Endonuclease 8-like 1 dsDNA Proliferating cell nuclear antigen DAMMIN model

Synchrotron SAXS data from solutions of Human NEI like DNA glycosylase 1 (NEIL1) bound to Proliferating Cell Nuclear Antigen (PCNA) and DNA in 25mM HEPES 100mM NaCl 1mM DTT, pH 7.5 were collected on the 12.3.1 (SIBYLS) camera at the Advanced Light Source (ALS) storage ring (Berkeley, CA, USA) using a Pilatus3 X 2M detector at a sample-detector distance of 1.4 m and at a wavelength of λ = 0.1127 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 0.6 and 2.8 mg/ml were measured at 10°C. 24 successive 0.200 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

We have attempted to characterize this novel complex using SAXS. This complex shows prominent concentration dependency. Due to presence of DNA as part of this complex, "280nm UV absorbance"-based concentration estimations have been largely compromised, producing un-interpretable forward scattering upon concentration normalization. Thus we have relied upon the "concentration-independent" SAXS-MoW to estimate the MW of the scatterer at physiologically relevant micromolar concentrations. MW estimated using qmax=0.2431 (<=8/Rg) suggests this novel complex to be 86.7kDa, comparable to theoretical value of 85.5 kDa.

Endonuclease 8-like 1
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   44.7 kDa
 
UniProt   Q96FI4
Sequence   FASTA
 
dsDNA
Mol. type   DNA
Olig. state   Monomer
Mon. MW   2.4 kDa
Sequence   FASTA
 
Proliferating cell nuclear antigen
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   29.7 kDa
 
UniProt   P12004
Sequence   FASTA