DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

The MmpL family of proteins translocates complex (glyco)lipids and siderophores across the cell envelope of mycobacteria and closely related Corynebacteriaceae, and plays important roles in the biogenesis of the outer membrane of these organisms. Despite their significance in the physiology and virulence of Mycobacterium tuberculosis, and from the perspective of developing novel antituberculosis agents, little is known about their structure and mechanism of translocation. In this study, the essential mycobacterial mycolic acid transporter, MmpL3, and its ortholog in Corynebacterium glutamicum, CmpL1, were investigated as prototypical MmpL proteins to gain insight into the transmembrane topology, tertiary and quaternary structures, and functional regions of this transporter family. Our combined genetic, biochemical and biophysical studies indicate that MmpL3 and CmpL1 are structurally similar to Gram-negative Resistance-Nodulation and Division efflux pumps. They harbor twelve transmembrane segments interrupted by two large soluble periplasmic domains, and function as homotrimers to export long-chain (C22-C90) mycolic acids, possibly in their acetylated form, esterified to trehalose. The mapping of a number of functional residues within the middle region of the transmembrane domain of MmpL3 shows a striking overlap with mutations associated with resistance to MmpL3 inhibitors. Our results suggest that structurally diverse inhibitors of MmpL3 all target the proton translocation path of the transporter and that multi-resistance to these inhibitors is enabled by conformational changes in MmpL3.

The structure of fructose 1,6-bisphosphate aldolase shows three distinct modes of product binding that are correlated to the disposition of the C-terminal region and depicts a possible trajectory for product exchange. The structure also indicates binding preference for monobasic triose phosphates.

The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4-derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refrned phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound fl-barrel of the parallel a/fl class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other ,8-barrel structures, the known active-site residues are located in the center of the ,8-barrel and are accessible to substrate from the COOH side of the fl-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the fl-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.Aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) is an ubiquitous and abundant glycolytic enzyme that plays a central and pivotal role in glycolysis and fructose metabolism. Aldolases from all species catalyze the reversible aldol cleavage of fructose 1,6-bisphosphate (Fru-1,6-P2) into the triose phosphates, Dglyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis proceeds by two distinct chemical pathways in aldolases. In class I aldolases, found in plants and higher animals, catalysis depends upon Schiff-base formation with the substrate (1), whereas in class II aldolases, found mostly in molds and bacteria, catalysis requires a metal cofactor such as Zn2+ (2). Demonstrable activity by aldolases also exists toward substrates such as fructose 1-phosphate (Fru-1-P), and the differential activity by aldolases toward Fru-1,6-P2 and Fru-1-P has been used as a basis to discriminate between the various isozymes in vertebrates (3). In rabbit tissues, aldolase A has been isolated from muscle, aldolase B from liver, and aldolase C from brain. The three forms have been purified to homogeneity and extensively characterized (3-5). The enzymes have a relative molecular mass (Mr) of approximately 158,000 and a tertiary structure composed of...

The crystal structure of p24, the single-stranded DNA (ssDNA) binding subunit of the plant defense transcription factor PBF-2, has been determined to 2.3 A resolution. p24 is representative of a novel family of ubiquitous plant-specific proteins that we refer to as the Whirly family because of their quaternary structure. PBF-2 is composed of four p24 molecules that interact through a helix-loop-helix motif. This interaction produces a central pore, with beta-strands radiating outwards, resulting in a whirligig appearance to the quaternary structure. The noncrystallographic C(4) symmetry arrangement of p24 subunits is novel for ssDNA binding proteins and may explain the binding specificity of PBF-2. This structural arrangement also supports the role of PBF-2 in binding melted promoter regions to modulate gene expression.