RAD50 and NBS1 form a stable complex functional in DNA binding and tethering

Abstract

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.

Figure 1.

(A) Coomassie stained SDS-PAGE gel of purified protein preparations. Lane 1, molecular size marker (m; molecular mass indicated in kilo Dalton); lane 2, RAD50/MRE11 complex (RM); lane 3, RAD50/MRE11/NBS1 complex (RMN); lane 4, RAD50/NBS1 complex (RN). (B) Immuno-blotting analysis of purified proteins. Blots containing purified RM, RMN and RN preparations were probed with antibodies directed against RAD50, MRE11 and NBS1, as indicated.

Figure 2.

SFM analysis of RM (A), RMN (B) and RN preparations (C). Purified protein was deposited on mica and imaged by tapping mode SFM at 1 μm × 1 μm scale in air (left panels). The scale bars are 100 nm. The colour bars represent the height from 0 to 3 nm (brown to pink). The right panels are examples of individual complexes enlarged and presented as surface plots (0.105 μm × 0.105 μm scale) in which RAD50 has a monomeric (I), dimeric (II) or multimeric (III) stoichiometry.

Figure 3.

The volume distributions of the globular part for RM (A), RMN (B) and RN (C) are presented in histograms. Purified protein was deposited on mica and imaged by tapping mode SFM at 1 μm × 1 μm scale in air. From such images isolated dimeric RAD50 complexes were selected for volume determination of the globular part. The x-axis is the relative molecular volume obtained from the SFM data, the y-axis is the number of protein molecules in each peak. A Gaussian distribution was calculated for the data and is displayed as a solid black line. The average volumes estimated from the Gaussian distributions are shown above each peak.

Figure 4.

DNA binding by RM, RMN and RN. (A) Alexa Fluor 532 labeled dsDNA66 (1 nM), was incubated with RM (37, 75, 125, 250, 500 and 1000 ng), RMN (idem) or RN (2.3, 4.5, 9, 18, 37, 75, 125 and 250 ng) for 20 min at 25°C in a final volume of 20 μl. Complexes formed were separated by 5% non-denaturing PAGE and visualized by fluorescence scanning. FD, free DNA. (B) Free DNA was quantified and plotted against the amounts of protein added.

Figure 5.

DNA tethering by RM (A), RMN (B) and RN (C). Reaction mixtures containing 15 nM of 3.0-kb linear DNA fragment and 25 nM of purified protein were deposited on mica and imaged by tapping mode SFM. The scale bars are 200 nm. Colour represents height from 0 to 3 nm (brown to pink), as shown by the inserts. DNA tethers (triangle), free DNA molecules (star) and DNA molecules involved in DNA tethering (circle) were all quantified as described in ‘Materials and Methods’ section.