Isolation and characterization of monomeric human RAD51: a novel tool for investigating homologous recombination in cancer

Rinaldi F, Schipani F, Balboni B, Catalano F, Marotta R, Myers S, Previtali V, Veronesi M, Scietti L, Cecatiello V, Pasqualato S, Ortega J, Girotto S, Cavalli A, Angewandte Chemie International Edition (2023) DOI

SASDQU9 – His-Tagged Monomeric DNA repair protein RAD51 homolog 1 double mutant [F86E, A89E] in complex with His-tagged fourth BRC repeat (BRC4)

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged)
Breast cancer type 2 susceptibility protein

MW^experimental	45	kDa
MW^expected	46	kDa
V^Porod	78	nm³

log I(s) 4.67×10^-2 4.67×10^-3 4.67×10^-4 4.67×10^-5

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein small angle scattering data

s, nm^-1

Log plot Log-Log plot

ln I(s)

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein Guinier plot

ln 4.68×10^-2 R_g: 3.0 nm 0 (3.0 nm)^-2 s²

(sR_g)²I(s)/I(0)

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein Kratky plot

1.104 0 √3 sR_g

p(r)	R_g: 3.4 nm 0 D_max: 16.5 nm

Data validation

Experimental data range

p(r) fit to data

Metric

Value

s_min D_max / π

0.8

N_Shannon

Worse

Better

Metric

Value

p_cormap

χ²

0.144
1.159

Worse

Better

Fits and models

log I(s)	s, nm^-1
+3 Δ/σ -3	s, nm^-1

His-Tagged Monomeric DNA repair protein RAD51 homolog 1 double mutant [F86E, A89E] in complex with His-tagged fourth BRC repeat (BRC4) Rg histogram

R_g, nm

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein EOM/RANCH model

log I(s)	s, nm^-1
+3 Δ/σ -3	s, nm^-1

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein ALPHAFOLD model

log I(s)	s, nm^-1
+3 Δ/σ -3	s, nm^-1

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) Breast cancer type 2 susceptibility protein PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from solutions of His-Tagged DNA repair protein RAD51 homolog 1 [F86E, A89E] in complex with the His-tagged fourth BRC repeat of BRC4 in 20 mM HEPES, 200 mM Na2SO4, 5% glycerol, 0.1 mM EDTA, pH 8 were collected on the B21 beam line at the Diamond Light Source (Didcot, UK) using a Eiger 4M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.095373 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 100.00 μl sample at 6.1 mg/ml was injected at a 0.07 ml/min flow rate onto a GE Superdex 200 Increase 3.2/300 column at 15°C. 36 successive frames were collected through the sample elution peak (from a total of 599 frames). The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Experimental molecular weight has been obtained through static light scattering (SLS).

DNA repair protein RAD51 homolog 1 (F86E, A89E, His-Tagged) (HisRAD51 [F86E,A89E])
Mol. type		Protein
Organism		Homo sapiens
Olig. state		Monomer
Mon. MW		39.8 kDa

UniProt		Q06609 (1-339)
Sequence		FASTA

Breast cancer type 2 susceptibility protein (HisTaggedBRC4)
Mol. type		Protein
Organism		Homo sapiens
Olig. state		Monomer
Mon. MW		6.3 kDa

UniProt		P51587 (1517-1551)
Sequence		FASTA

PDB ID		1N0W