Shin Ichiro Yonekura

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca2+-ATPase in the absence of Ca2+

Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca2+-ATPase, a representative P-type ATPase, in the absence of Ca2+ with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca2+-ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca2+-ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of “superfluorescence.” Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.

Purification and characterization of Caenorhabditis elegans NTH, a homolog of human endonuclease III: essential role of N-terminal region.

Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast searching of the Wormbase database retrieved a sequence R10E4.5, highly homologous to the human NTH1. However, the R10E4.5-encoded protein did not have NTH activity, and this was considered to be due to lack of the N-terminal region crucial for the activity. Therefore, we purified the protein encoded by the sequence containing both R10E4.5 and the 117-bp region upstream from it, and found that the protein had the NTH activity. The endogenous CeNTH in the extract of C. elegans showed the same DNA glycosylase activity. Therefore, we concluded that the genuine C. elegans NTH gene is not the R10E4.5 but the sequence containing both R10E4.5 and the 117-bp upstream region. NTH-deficient C. elegans showed no difference from the wild-type in lifespan and was not more sensitive to two oxidizing agents, H2O2 and methyl viologen. This suggests that C. elegans has an alternative DNA glycosylase that repairs pyrimidine bases damaged by these agents. Indeed, DNA glycosylase activity that cleaved thymine glycol containing oligonucleotides was detected in the extract of the NTH-deficient C. elegans.

Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans

Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.

Error-Prone Translesion DNA Synthesis by Escherichia coli DNA Polymerase IV (DinB) on Templates Containing 1,2-dihydro-2-oxoadenine.

Escherichia coli DNA polymerase IV (Pol IV) is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS) are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg), 5-formyluracil (5-fodU), 5-hydroxymethyluracil (5-hmdU), 7,8-dihydro-8-oxoguanine (8-oxodG) and 1,2-dihydro-2-oxoadenine (2-oxodA). Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IVs ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:T→G:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:T→G:C mutations in E. coli, while the increase was significantly greater in E. coli overexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:T→G:C mutations.

KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/ AP lyase activity to prevent mutations in Escherichia coli

The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a β-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.

Functional analysis of SERCA1b, a highly expressed SERCA1 variant in myotonic dystrophy type 1 muscle

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca(2+) from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca(2+) uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca(2+) was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca(2+) homeostasis.

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.

Corrigendum to: Mn2+ transport by Ca2+-ATPase of sarcoplasmic reticulum (FEBS Letters, 590, 14, (2086-2095), 10.1002/1873-3468.12244)

Mn transport by Ca-ATPase of sarcoplasmic reticulum Shin-Ichiro Yonekura and Chikashi Toyoshima This article corrects: Mn transport by Ca-ATPase of sarcoplasmic reticulum, Volume 590, Issue 14, 2086– 2095. Article first published on 23 June 2016. DOI:10.1002/1873-3468.12244 A figure was accidentally duplicated and appears in the article as both Figures 4 and 5. The correct Figure 4 is shown below:

Mn2+ transport by Ca2+‐ATPase of sarcoplasmic reticulum

Ca2+‐ATPase of sarcoplasmic reticulum is known to pump Mn2+ in addition to Ca2+, but whether its transport mechanism is identical to that of Ca2+ is ambiguous. To clarify, we examined, by atomic absorption spectroscopy, competition between Mn2+ and Ca2+ in active transport using vesicles of sarcoplasmic reticulum (SR). Here, we demonstrate that Ca2+‐ATPase transports Ca2+ and Mn2+ concomitantly but has a much lower affinity for Mn2+ (apparent Kd ~ 0.5 mm). Stoichiometries of transported ions per ATP hydrolysed, Vmax values and activation energies are very similar. Altogether, Ca2+‐ATPase appears to use the same mechanism for transporting Mn2+ as that for Ca2+.