PDBsum entry 1mje

Gene regulation/antitumor protein/DNA

PDB id: 1mje

Contents

Protein chains

42 a.a.

600 a.a. *

DNA/RNA

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Brca2 function in DNA binding and recombination from a brca2-Dss1-Ssdna structure.

Authors

H.Yang, P.D.Jeffrey, J.Miller, E.Kinnucan, Y.Sun, N.H.Thoma, N.Zheng, P.L.Chen, W.H.Lee, N.P.Pavletich.

Ref.

Science, 2002, 297, 1837-1848. [DOI no: 10.1126/science.297.5588.1837]

PubMed id

12228710

Abstract

Mutations in the BRCA2 (breast cancer susceptibility gene 2) tumor suppressor lead to chromosomal instability due to defects in the repair of double-strand DNA breaks (DSBs) by homologous recombination, but BRCA2's role in this process has been unclear. Here, we present the 3.1 angstrom crystal structure of a approximately 90-kilodalton BRCA2 domain bound to DSS1, which reveals three oligonucleotide-binding (OB) folds and a helix-turn-helix (HTH) motif. We also (i) demonstrate that this BRCA2 domain binds single-stranded DNA, (ii) present its 3.5 angstrom structure bound to oligo(dT)9, (iii) provide data that implicate the HTH motif in dsDNA binding, and (iv) show that BRCA2 stimulates RAD51-mediated recombination in vitro. These findings establish that BRCA2 functions directly in homologous recombination and provide a structural and biochemical basis for understanding the loss of recombination-mediated DSB repair in BRCA2-associated cancers.

Figure 3.
Fig. 3. DSS1 binds BRCA2 in an extended conformation, interacting with basic grooves on the helical domain, OB1 and OB2 domains. (A) The NH[2]-terminal segment of DSS1 (orange) tunnels through the helical domain (magenta), and also contacts OB1 (green). View is rotated ~180° about the horizontal axis relative to Fig. 1D. In addition to the contacts discussed in the text, Glu14 of DSS1 interacts with Lys2712 of OB1, Glu15 with Gln2576, Asp16 with Lys2671, Asp17 with Lys2551, and Glu18 with Lys2435 and Arg2441. In addition, Asp16 forms a salt bridge with Arg57, located on the COOH-terminal DSS1 segment that binds on the opposite side of BRCA2. (B) Surface representation of the helical domain-OB1 region that binds the NH[2]-terminal segment of DSS1, in an orientation similar to that of (A). The surface is colored according to the electrostatic potential (-30 kT to +30 kT), calculated in the absence of DSS1 with the program GRASP (92). The backbone traces of the NH[2]-terminal and part of the COOH-terminal DSS1 segments are shown as white coils, and the side chains, in yellow. The white-dotted line indicates the 11-residue disordered segment between the NH[2]- and COOH-terminal segments of DSS1. DSS1 residues that are entirely buried below the surface of BRCA2 are indicated in parenthesis. The side chains for residues 46 to 49 of the COOH-terminal segment have poor electron density and are omitted. (C) Surface representation of the helical domain-OB1-OB2 region that binds the COOH-terminal segment of DSS1, in an orientation rotated ~90° about the vertical axis relative to (B). Hydrophobic interactions include the DSS1 Phe^19, Trp39, Trp43, which pack with Trp2646, Phe^2722, and with an opening in the hydrophobic core of OB1, respectively. In addition, the majority of the acidic DSS1 residues are near clusters of basic BRCA2 residues. These include Glu40, Asp41, and Asp44, which are near Arg2708, Lys2898, and Lys2655; Asp50 and Asp51, which are near Arg2708 and Arg2451; and Glu20 and Glu21 [from the NH[2]-terminal segment in (B)] which are near Lys2712, Arg2717, Arg2590 and Lys2595.

Figure 4.
Fig. 4. BRCA2 contains a three-helix bundle (3HB) similar to the helix-turn-helix dsDNA binding motif. The 35-residue BRCA2 3HB is superimposed on the 36-residue 3HB domain of the Hin recombinase DNA binding domain (48). The 35 residues correspond to the entire 3HB, starting with the NH[2]-terminus of T 2 and ending at the COOH-terminus of T 4. There is only one residue insertion between H2 and H3 of the Hin recombinase 3HB in the alignment.

The above figures are reprinted by permission from the AAAs: Science (2002, 297, 1837-1848) copyright 2002.