The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.

Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an ϳ16-Å structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture (ϳ5.6 subunits/turn, 24-Å helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems.

Germ-line mutations in BRCA2 account for approximately half the cases of autosomal dominant familial breast cancers. BRCA2 has been shown to interact directly with RAD51, an essential component of the cellular machinery for homologous recombination and the maintenance of genome stability. Interactions between BRCA2 and RAD51 take place by means of the conserved BRC repeat regions of BRCA2. Previously, it was shown that peptides corresponding to BRC3 or BRC4 bind RAD51 monomers and block RAD51-DNA filament formation. In this work, we further analyze these interactions and find that at lower molar ratios BRC3 or BRC4 actually bind and form stable complexes with RAD51-DNA nucleoprotein filaments. Only at high concentrations of the BRC repeats are filaments disrupted. The specific protein-protein contacts occur in the RAD51 filament by means of the N-terminal domain of RAD51 for BRC3 and the nucleotide-binding core of RAD51 for BRC4. These observations show that the BRC repeats bind distinct regions of RAD51 and are nonequivalent in their mode of interaction. The results provide insight into why mutation in just one of the eight BRC repeats would affect the way that BRCA2 protein interacts with the RAD51 filament. Disruption of a single RAD51 interaction site, one of several simultaneous interactions occurring throughout the BRC repeat-containing exon 11 of BRCA2, might modulate the ability of RAD51 to promote recombinational repair and lead to an increased risk of breast cancer.electron microscopy ͉ nucleoprotein filaments ͉ recombination ͉ genome stability and dynamics ͉ structural biology M utations in BRCA2 are associated with an increased risk of breast and ovarian cancers, and almost half the cases of inherited early onset breast cancers have been linked to mutations in BRCA2. The product of this gene, BRCA2 protein (384 kDa), interacts with RAD51, and it has been shown that both proteins are essential for homologous recombination, DNA repair, and the maintenance of genome stability. There appear to be a number of different interactions between BRCA2 and RAD51. One such interaction involves a C-terminal region of BRCA2 (1). In addition, BRCA2 interacts with RAD51 through the eight conserved BRC repeats in BRCA2 (2, 3), and mutations within these repeats are associated with cancer predisposition. The deletion of several BRC repeats in mice leads to cancer (4), and somatic mutations in BRC repeats have been found to be associated with breast cancer (5). Although there are eight BRC repeats in human BRCA2, in other organisms the number of BRC repeats is quite variable, with 15 found in a Trypanosoma BRCA2-like protein (6) and only one found in the Ustilago Brh2 protein (7).Many details of the interactions between BRCA2 and RAD51 are unclear, such as whether BRCA2 binds to the N-terminal domain of RAD51 (8) or to the nucleotide-binding core (3, 9). Recently, a fusion protein containing the nucleotide-binding core of RAD51 and a single BRC repeat was crystallized. Analysis of the x-ray structure of the fusion led t...

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from (sea anemones, jellyfish), the most primitive animals with muscles, and (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba showed a similar, but modified, version of the motif, while the amoeba and fission yeast () showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.