Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

Abstract

17Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) 18 protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and 19Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along 20 with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of 21 PALB2 DNA binding and its function are poorly understood. We identified a major DNA-binding site 22 4.0±1.3 nM to 316±59 nM in the case of T1 and from 4.8±0.4 nM to 167±50 nM in the case of PB2-129 573 (Fig. 2). DNA binding was moderately affected by mutations of two other clusters, including 130 K45A/K50A, for which the Kd was increased to 28±5.2 nM (Fig. S4), and the triple mutant 131 R170A/K174A/R175A with similar change in Kd. From these experiments, we concluded that the main 132 DNA binding site is formed by residues 146-149 with a potential minor contribution from other basic 133 amino acids of the N-DBD. 134 135 Figure 2. Mutation of DNA binding residues. Isotherm of fluorescence anisotropy of FAM-ss49 (5 136 nM) titrated by PALB2 T1 (blue, open circles) and PB2-573 (magenta, open triangles) fragments and 137 their mutants: T1 146-RK/AA (filled blue circles), T1 146-RRKK/AAAA (crossed open blue circles), 138and 573 146-RRKK/AAAA (filled magenta triangles) under conditions identical to those in Fig. 1. 139 Figure S4. DNA binding of T1 mutants. Equilibrium binding of 5 nm D) to PALB2 T1 (blue). In A) and B) isotherm for K45A is in green, for K50A in red, for K45A/K50A 141 in black. In C) and D) isotherm for R170A is in red, for K174A in green, for R170A/K174A in magenta, 142 for R170A/R175A in orange, and for R170A/K174A/R175A in black. The binding buffer is 20 mM Tris-143 acetate pH 7.0, 100 mM NaCl, 5% glycerol, 10% DMSO. 144