The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50[middle dot]hMRE11[middle dot]NBS1 complex DNA repair activity

NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.     

The sre gene (ORF469) encodes a site-specific recombinase responsible for integration of the R4 phage genome.

The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Isolation and identification of a humanTRPV1 activating compound from soy sauce

Transient receptor potential vanilloid 1 (TRPV1) was identified as a receptor of capsaicin, which is a pungent ingredient in hot red peppers. Due to its relevance for nociception, a physiological and pharmacological study of TRPV1 has also been developed. Therefore, it is important to enrich scientific knowledge regarding the TRPV1 activating or inhibiting compounds. In this study, we fractionated soy sauce based on the human TRPV1 (hTRPV1) activity using column chromatography and purified 5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (perlolyrine) as an hTRPV1-activating compound. Additionally, perlolyrine activates the human transient receptor potential ankyrin 1 (hTRPA1). The EC₅₀ of hTRPV1 and hTRPA1 were 2.87 and 1.67 μmol L^?1, respectively. HPLC quantification of soy sauces showed that they contain 2.22–12.13 μmol L^?1 of perlolyrine. The sensory evaluation revealed that perlolyrine has taste modification effect. The results of this study, for the first time, suggest that perlolyrine induces the activation of hTRPV1 and hTRPA1.     

Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.     

Interruptions in the expanded ATTCT repeat of spinocerebellar ataxia type 10: repeat purity as a disease modifier?

Spinocerebellar ataxia type 10 (SCA10) is one of numerous genetic disorders that result from simple repeat expansions. SCA10 is caused by expansion of an intronic ATTCT pentanucleotide repeat tract. It is clinically characterized by progressive ataxia, seizures, and anticipation, which can vary within and between families. We report two SCA10 families showing distinct frequencies of seizures and correlations of repeat length with age at onset. One family displayed uninterrupted ATTCT expansions, whereas the other showed multiple interruptions of the repeat by nonconsensus repeat units, which differed both in the length and/or sequence of the repeat unit. Disease-causing microsatellite expansions have been assumed to be composed of uninterrupted pure repeats. Our findings for SCA10 challenge this convention and suggest that the purity of the expanded repeat element may be a disease modifier.