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DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.

Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices. Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged. We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position. The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined. The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes.

Studies were conducted on 2 cows chronically infected with bluetongue virus (BTV) acquired in utero from their dam. In previous research, BTV had been isolated 4 times from 1 cow and 8 times from the other. BTV was undetectable between spontaneous febrile and leukopenic episodes and antibodies to BTV were not detectable in the serum. Previous work had shown that bites by uninfected females of the vector Culicoides variipennis (Coquillett) caused virus to recrudesce in the blood to detectable levels in a bull with a similar BTV infection. After 1 of the 2 cows became hypersensitive to the bites of the vector, the cows were inoculated with homogenized salivary glands from the vector in an effort to increase the viral concentration to detectable levels. Bites by uninfected vectors were used 9.5-48 hr after inoculation of salivary glands in attempts to recover BTV biologically. In 2 of 6 experiments, after inoculations of the glands, BTV was isolated and confirmed from some of the blood samples and from some pools of C. variipennis females that had blood fed on both animals.

A summary of the more important concerns of the Working Team with particular reference to bluetongue (BT) virus (BTV), is as follows: With the exception of Australia, the US, and possibly parts of Africa, there are almost no concrete data that could be used to direct control efforts against the responsible vector(s) of BTV in a specific geographic area. In Australia, a broad plan of attack yielded data that showed that members of the subgenus Avaritia are the primary vector species. It has been clearly shown that Culicoides variipennis (Coq.) is the dominant vector species for most of the US. In the Middle East and certain areas of Africa, isolations from possible vector species indicated that C. imicola K., again probably a species complex and belonging to the subgenus Avaritia, is one of the likely vectors. Except for some subgenera, the taxonomy for much of the Culicoides remains in disarray. The subgenus Oecacta for example still remains a catchall for many species. Although a number of proposals have been made for delineating the species of the C. (Monoculicoides) variipennis species complex, none have been initiated because of the lack of funds. The Working Team strongly feels that the lack of definition of important species complexes is the result of short-term planning that does not consider the fact that efficiency can be increased and a great deal of monies saved by directing control toward the specific species or populations thereof that are responsible for a problem.(ABSTRACT TRUNCATED AT 250 WORDS)