Tatsushi Ruike

TRF4 Is Involved in Polyadenylation of snRNAs in Drosophila melanogaster

ABSTRACT The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3′-5′ exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.

DmGEN shows a flap endonuclease activity, cleaving the blocked‐flap structure and model replication fork

Drosophila melanogaster XPG‐like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2/XPG family. The DmGEN protein has two nuclease domains (N and I domains) similar to XPG/class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN‐1/class II and EXO‐1/class III nucleases. To confirm the properties of DmGEN, we characterized the active‐site mutant protein (E143A E145A) and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick‐dependent 5′−3′ exonuclease activity. Unlike XPG, DmGEN could not incise the bubble structure. Interestingly, based on characterization of flap endonuclease activity, DmGEN preferred the blocked‐flap structure as a substrate. This feature is distinctly different from FEN‐1. Furthermore, DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus, our studies revealed that DmGEN belongs to a new class (class IV) of the RAD2/XPG nuclease family. The biochemical properties of DmGEN and its possible role are also discussed.

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase δ and εin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.

Drosophila DNA Polymerase ζ Interacts with Recombination Repair Protein 1, the Drosophila Homologue of Human Abasic Endonuclease 1

Abasic (AP) sites are a threat to cellular viability and genomic integrity, since they impede transcription and DNA replication. In mammalian cells, DNA polymerase (pol) β plays an important role in the repair of AP sites. However, it is known that many organisms, including Drosophila melanogaster, do not have a pol β homologue, and it is unclear how they repair AP sites. Here, we screened for DNA polymerases that interact with the Drosophila AP endonuclease 1 homologue, Rrp1 (recombination repair protein 1), and found that Drosophila pol ζ (Dmpol ζ), DmREV3 and DmREV7 bound to Rrp1 in a protein affinity column. Rrp1 directly interacted with DmREV7 in vitro and in vivo but not with DmREV3. These findings suggest that the DNA polymerase partner for Rrp1 is Dmpol ζ and that this interaction occurs through DmREV7. Interestingly, DmREV7 bound to the N-terminal region of Rrp1, which has no known protein homologue, suggesting that this binding is a species-specific event. Moreover, DmREV7 could stimulate the AP endonuclease activity of Rrp1, but not the 3′-exonuclease activity, and form a homomultimer. DmREV3 could not incorporate nucleotides at the 5′-incised tetrahydrofran sites but did show strand displacement activity for one-nucleotide-gapped DNA, which was not influenced by either DmREV7 or Rrp1. Methyl methanesulfonate and hydrogen peroxide treatments increased mRNA levels of DmREV3 and DmREV7. On the basis of the direct interaction between DmREV7 and Rrp1, we suggest that Dmpol ζ may be involved in the repair pathway of AP sites in DNA.

Promotion of crystalline cellulose degradation by expansins from Oryza sativa

Main conclusionEnzymatic activities ofOryza sativaexpansins, which were heterologously overexpressed inEscherichia coli, were analyzed. Results suggested that expansins promote degradation of cellulose by cellulase in a synergistic manner.AbstractSustainable production of future biofuels is dependent on efficient saccharification of lignocelluloses. Expansins have received a lot of attention as proteins promoting biological degradation of cellulose using cellulase. The expansins are a class of plant cell wall proteins that induce cell wall loosening without hydrolysis. In this study, the expansins from Oryza sativa were classified using phylogenetic analysis and five proteins were selected for functional evaluation. At low cellulose loading, the cellulase in expansin mixtures was up to 2.4 times more active than in mixtures containing only cellulase, but at high cellulose loading the activity of cellulase in expansin mixtures and cellulase only mixtures did not differ. Furthermore, expansin activity was greater in cellulase mixtures compared with cellulase-deficient mixtures. Therefore, the expansins showed significant synergistic activity with cellulase. Expansin may play an important role in efficient saccharification of cellulose.

Enhancement of Cellulose Degradation by Cattle Saliva

Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale.

Distribution and metabolism of 14C-Sulfoquinovosylacylpropanediol (14C-SQAP) after a single intravenous administration in tumor-bearing mice

Abstract Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.