Biology subjects

Ura, K.

Publications and source records attributed to Ura, K..

5 recordsLinked to original sources

bioRxiv2024

Impact of an Oxidative RNA Lesion on in vitro Replication Catalyzed by SARS-CoV-2 RNA-dependent RNA Polymerase

The production of reactive oxygen species in response to RNA virus infection results in the oxidation of viral genomic RNA within infected cells. These oxidative RNA lesions undergo replication catalyzed by the viral replisome. G to U transversion mutations are frequently observed in the SARS-CoV-2 genome and may be linked to the replication process catalyzed by RNA-dependent RNA polymerase (RdRp) past the oxidative RNA lesion 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG). To better understand the mechanism of viral RNA mutagenesis, it is crucial to elucidate the role of RdRp in replicating across oxidative lesions. In this study, we investigated the RNA synthesis catalyzed by the reconstituted SARS-CoV-2 RdRp past a single 8-oxo-rG. The RdRp-mediated primer extension was significantly inhibited by 8-oxo-rG on the template RNA. During the blockage of the extension reaction, the rate of product release by RdRp was extremely slow, indicating that the RdRp machinery remained bound to the replicating primer/template RNA. Once RdRp was able to bypass 8-oxo-rG, it preferentially incorporated rCMP, with a lesser amount of rAMP opposite 8-oxo-rG. In contrast, RdRp demonstrated greater activity in extending from the mutagenic rA:8-oxo-rG terminus compared to the lower efficiency of extension from the rC:8-oxo-rG pair. Based on steady-state kinetic analyses for the incorporation of rNMPs opposite 8-oxo-rG and chain extension from rC:8-oxo-rG or rA:8-oxo-rG, the relative bypass frequency for rA:8-oxo-rG was found to be seven-fold higher than that for rC:8-oxo-rG. Therefore, the properties of RdRp indicated in this study may contribute to the mechanism of mutagenesis of the SARS-CoV-2 genome.

biochemistry↗

bioRxiv2023

DNA methylation signature in NSD2 loss-of-function variants appeared similar to that in Wolf-Hirschhorn syndrome

PurposeWolf-Hirschhorn syndrome (WHS), a contiguous gene syndrome caused by the hemizygous deletion of the distal short arm of chromosome 4 where NSD2 is, reportedly exhibits specific DNA methylation signatures in peripheral blood cells. However, responsible genomic loci for signatures are unreported. The objective of the study is to define the loci of WHS-related DNA methylation signatures and to explore the role of NSD2 for the signatures. MethodsWe conducted genome-wide methylation analysis of individuals with WHS or NSD2 variants using array. We studied genome-edited knock in mice or induced pluripotent stem cells to explore the function of NSD2 variants which are observed in congenital anomaly cases. ResultsThree undiagnosed cases with NSD2 variants showed WHS-related DNA methylation signatures. These variants were validated to be NSD2 loss-of-function in induced pluripotent stem cells or genome-edited knock-in mice. p.Pro905Leu variant decreased Nsd2 protein levels, and changed Histone H3-Lysine 36 demethylation levels in similar way in the same genomic regions as Nsd2 knock out mice regulated. Nsd2 knock out mice exhibited common DNA methylation changes. ConclusionThese results revealed that WHS-related DNA methylation signatures are dependent on NSD2 dysfunction and are useful in diagnosing NSD2 variants of unknown significance.

genetics↗

bioRxiv2019

Processing of a single ribonucleotide embedded into DNA by human nucleotide excision repair and DNA polymerase η

DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase {eta} (Pol {eta}) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol {eta} accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol {eta}. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol {eta} leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol {eta}, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.

genetics↗

bioRxiv2019

Quantification of the effect of site-specific histone acetylation on chromatin remodeling rate

Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II-, and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ~3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.

biophysics↗

bioRxiv2017

Changes in levels of major yolk protein in the coelomic fluid and gonad during the reproductive cycle in wild sea urchins, Mesocentrotus nudus

Both female and male sea urchins accumulate the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before gametogenesis, and MYP is the most abundant protein in the coelomic fluid of both sexes. In females, MYP in the coelomic fluid is taken up by the nutritive phagocytes and transported to the growing oocytes. This study examined quantitative changes of MYP in the coelomic fluid of both sexes during the reproductive cycle of wild sea urchins, Mesocentrotus nudus. Levels of MYP in the coelomic fluid of females increased and reached a peak at the histological pre-mature stage of gonad activity (i.e. Stage 3), and positive correlation between the MYP level and the gonadosomatic index (GSI) was observed. In male sea urchins the level of MYP in the coelomic fluid increased at the pre-mature stage, though positive correlation between the MYP level and GSI was not observed. These results indicate that MYP in the coelomic fluid is suitable as a biomarker of the onset and progression of sexual maturity in female sea urchins.

physiology↗