Expression, purification and crystallization of Swi5 and the Swi5-Sfr1 complex from fission yeast.

The assembly of the presynaptic filament of recombinases represents the most important step in homologous recombination. The formation of the filament requires assistance from mediator proteins. Swi5 and Sfr1 have been identified as mediators in fission yeast and these proteins form a complex that stimulates strand exchange. Here, the expression, purification and crystallization of Swi5 and its complex with an N-terminally truncated form of Sfr1 (DeltaN180Sfr1) are presented. Analytical ultracentrifugation of the purified samples showed that Swi5 and the protein complex exist as tetramers and heterodimers in solution, respectively. Swi5 was crystallized in two forms belonging to space groups C2 and R3 and the crystals diffracted to 2.7 A resolution. Swi5-DeltaN180Sfr1 was crystallized in space group P2(1)2(1)2 and the crystals diffracted to 2.3 A resolution. The crystals of Swi5 and Swi5-DeltaN180Sfr1 are likely to contain one tetramer and two heterodimers in the asymmetric unit, respectively.

Light-dependent mutagenesis by benzo[a]pyrene is mediated via oxidative DNA damage.

Benzo[a]pyrene (B[a]P) is an environmental carcinogenic polycyclic aromatic hydrocarbon (PAH). Mammalian enzymes such as cytochrome P-450s and epoxide hydrase convert B[a]P to reactive metabolites that can covalently bind to DNA. However, some carcinogenic compounds that normally require metabolic activation can also be directly photoactivated to mutagens. To examine whether B[a]P is directly mutagenic in the presence of light, we exposed Salmonella typhimurium strains with different DNA repair capacities to B[a]P and white fluorescent light at wavelengths of 370-750 nm. B[a]P plus light significantly enhanced the number of His+ revertants. Mutagenesis was completely light-dependent and required no exogenous metabolic activation. The order of mutability of strains with different DNA repair capacities was strain YG3001 (uvrB, mutMST) >> strain TA1535 (uvrB) > strain YG3002 (mutMST) > strain TA1975. The uvrB gene product is involved in the excision repair of bulky DNA adducts, and the mutMST gene encodes 8-oxoguanine (8-oxoG) DNA glycosylase, which removes 8-oxoG from DNA. Introduction of a plasmid carrying the mOgg1 gene that is the mouse counterpart of mutMST substantially reduced the light-mediated mutagenicity of B[a]P in strain YG3001. B[a]P plus light induced predominantly G:C --> T:A and G:C --> C:G transversions. We propose that B[a]P can directly induce bulky DNA adducts if light is present, and that the DNA adducts induce oxidative DNA damage, such as 8-oxoG, when exposed to light. These findings have implications for the photocarcinogenicity of PAHs.

Crystallization and preliminary X-ray analysis of the full-size cubic core of pig 2-oxoglutarate dehydrogenase complex.

The full-length (untruncated) dihydrolipoamide succinyltransferase from pig heart was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction patterns indicate that the crystal belongs to space group I432, with unit-cell parameter a = 189.9 A. The crystal structure has been preliminarily solved at 7 A resolution by the molecular-replacement method. The unit cell contains two cubic cores, in each of which 24 subunits of E2 are associated according to crystallographic 432 symmetry. At the corners of each cubic core, the catalytic domains of E2s form a trimer through tight interactions around the crystallographic threefold axes. In the electron-density maps, many small broad peaks are observed in regions expected to contain the remaining N-terminal domains (the E1/E3-binding domain and the lipoyl domain), suggesting flexibility of these domains relative to the core. The architecture of the cubic core is similar to that of the other truncated E2s. In the unit cell, however, the core-core contact occurs in a different direction from that found for the truncated proteins.