A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

DJ-1 family Maillard deglycases prevent acrylamide formation.

The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide formation at 100 °C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent acrylamide formation in food without depleting the components (asparagine and sugars) responsible for its formation.

Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.

An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study.

Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the Npro (EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed in Escherichia coli, Npro-fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM and Thermococcus kodakarensis Hef were prepared. We improved the protocol of refolding of Npro (EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.

Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

Control of enzyme reaction by a designed metal-ion-dependent α-helical coiled-coil protein.

Regulation of protein function by external stimuli is a fascinating target for de novo design. We have constructed a peptide that assembles into a homotrimer in the presence of metal ions, such as Ni(2+), Cu(2+), and Zn(2+). We fused the peptide construct to the DNA-binding domain (DBD) of the heat shock factor from Saccharomyces cerevisiae, which binds tandem repeats of the heat shock element (HSE). However, the fusion protein bound to the natural three tandem HSEs even in the absence of metal ions, although mainly as the dimerized protein. Using "skipped" HSEs containing six additional nucleotides inserted between two adjacent HSEs, to prevent interactions between the DBDs, we found the fusion protein bound to the new DNA target in a metal-ion-dependent manner, as monitored by a HindIII protection assay. The fusion protein containing two metal binding sites in the metal-ion-controlled domain inhibited RNA transcription by T7 RNA polymerase in the presence of metal ions, in a template containing skipped HSEs downstream of the T7 promoter. The designed protein therefore regulates the functions of the enzyme in a metal-ion-dependent manner.

The clamp-loading complex for processive DNA replication.

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC-PCNA-DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

Mutational analysis of Pyrococcus furiosus replication factor C based on the three-dimensional structure.

In eukaryotic DNA replication, replication factor C (RFC) acts as a "clamp loader" that loads PCNA onto a primed DNA template in an ATP-dependent manner. Proteins with functions essentially identical to that of RFC exist in Archaea. We have determined the crystal structure of the small subunit (RFCS) of Pyrococcus furiosus RFC at 2.8-A resolution. Using the information from the determined tertiary structure, we prepared several mutations in RFCS and biochemically characterized them. Truncation of the C-terminal alpha-helix (alpha16) causes a failure in RFCS oligomerization and a loss of the stimulating activity for the PCNA-dependent DNA synthesis by DNA polymerases. The site-directed reduction of the negative charges at the center part of the RFCS complex affected the stability of the RFC-PCNA interaction and reduced the clamp-loading activity. These results contribute to our general understanding of the structure-function relationship of the RFC molecule for the clamp-loading event.