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Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken Blymphocyte line DT40. REV1 ؊/؊ REV3 ؊/؊ REV7 ؊/؊ cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1؊/؊ cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Pol (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.Homologous DNA recombination (HR) is associated with various cell functions. It is involved in the repair of DNA double-strand breaks (DSBs), which may be caused by ionizing radiation (IR) or DNA interstrand-cross-linking agents, and which may lead to cell death if left unrepaired. HR also functions as a part of postreplication repair (PRR), avoiding DNA replication blocks induced by various endogenous or environmental genotoxic stresses. Mating-type gene switching in yeast and diversification of the immunoglobulin gene (Ig) in the immune cells of some vertebrate species are partly made through gene conversion events mediated by HR (15,37). In germ cells, HR is crucial to meiotic recombination, the process that increases genetic variation within a species (18).In yeast, the major components of PRR are divided roughly into two groups: translesion DNA synthesis (TLS) involving the Rad6-Rad18 epistasis group and HR composed of the Rad52 epistasis group (reviewed in references 4 and 10). TLS allows tolerance of DNA damage, employing a number of specialized DNA polymerases that are able to synthesize directly across template DNA lesions (reviewed in references 13 and 17). Vertebrate TLS polymerases have been reported to have biochemical functions basically similar to those of their yeast homologs. However, the biological role of the TLS polymerases is complicated and poorly characterized in vertebrate cells. Similarly, DNA polymerases involved in HR are only poorly understood even in yeast (reviewed in reference 16).REV genes were originally identified as responsible genes for reversible mutation of UV light-induced mutagenesis in the yeast Saccharomyces cerevisiae (21,23). Longstanding work in yeast and recent studies in vertebrates have revealed that Rev1 is one of the Y-family DNA polymerases (34), having deoxycytidyl transferase activity (31), and that Rev3 and Rev7 are the catalytic and regulatory subunits of DNA polymerase (Pol) capable of bypassing a UV-induced thymidine dimer (32; reviewed in references 20 and 30). The three ...

Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a fulllength PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.