RESUMO
Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes â¼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Animais , DNA/química , DNA de Cadeia Simples/genética , Humanos , Ácidos Nucleicos , Fases de Leitura Aberta , Ribonucleoproteínas/metabolismoRESUMO
Accounting for continual evolution of deleterious L1 retrotransposon families, which can contain hundreds to thousands of members remains a major issue in mammalian biology. L1 activity generated upwards of 40% of some mammalian genomes, including humans where they remain active, causing genetic defects and rearrangements. L1 encodes a coiled coil-containing protein that is essential for retrotransposition, and the emergence of novel primate L1 families has been correlated with episodes of extensive amino acid substitutions in the coiled coil. These results were interpreted as an adaptive response to maintain L1 activity, however its mechanism remained unknown. Although an adventitious mutation can inactivate coiled coil function, its effect could be buffered by epistatic interactions within the coiled coil, made more likely if the family contains a diverse set of coiled coil sequences-collectively referred to as the coiled coil sequence space. Amino acid substitutions that do not affect coiled coil function (i.e., its phenotype) could be "hidden" from (not subject to) purifying selection. The accumulation of such substitutions, often referred to as cryptic genetic variation, has been documented in various proteins. Here we report that this phenomenon was in effect during the latest episode of primate coiled coil evolution, which occurred 30-10 MYA during the emergence of primate L1Pa7-L1Pa3 families. First, we experimentally demonstrated that while coiled coil function (measured by retrotransposition) can be eliminated by single epistatic mutations, it nonetheless can also withstand extensive amino acid substitutions. Second, principal component and cluster analysis showed that the coiled coil sequence space of each of the L1Pa7-3 families was notably increased by the presence of distinct, coexisting coiled coil sequences. Thus, sampling related networks of functional sequences rather than traversing discrete adaptive states characterized the persistence L1 activity during this evolutionary event.
Assuntos
Evolução Molecular , Elementos Nucleotídeos Longos e Dispersos/genética , Primatas/genética , Retroelementos/genética , Sequência de Aminoácidos/genética , Animais , Análise Mutacional de DNA , Humanos , Mutação/genética , ProteínasRESUMO
Long interspersed nuclear element 1 (LINE-1 or L1) is the dominant retrotransposon in mammalian genomes. L1 encodes two proteins ORF1p and ORF2p that are required for retrotransposition. ORF2p functions as the replicase. ORF1p is a coiled coil-mediated trimeric, high affinity RNA binding protein that packages its full- length coding transcript into an ORF2p-containing ribonucleoprotein (RNP) complex, the retrotransposition intermediate. ORF1p also is a nucleic acid chaperone that presumably facilitates the proposed nucleic acid remodeling steps involved in retrotransposition. Although detailed mechanistic understanding of ORF1p function in this process is lacking, recent studies showed that the rate at which ORF1p can form stable nucleic acid-bound oligomers in vitro is positively correlated with formation of an active L1 RNP as assayed in vivo using a cell culture-based retrotransposition assay. This rate was sensitive to minor amino acid changes in the coiled coil domain, which had no effect on nucleic acid chaperone activity. Additional studies linking the complex nucleic acid binding properties to the conformational changes of the protein are needed to understand how ORF1p facilitates retrotransposition.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Fases de Leitura Aberta/genética , Animais , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/química , Humanos , CinéticaRESUMO
Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution.
Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Multimerização Proteica , Proteínas/química , Proteínas/metabolismo , DNA de Cadeia Simples , Humanos , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas/genéticaRESUMO
Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for â¼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/química , Elementos Nucleotídeos Longos e Dispersos , Fases de Leitura Aberta , Peptidilprolil Isomerase/química , Retroelementos/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatografia Líquida , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Insetos , Dados de Sequência Molecular , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Fosforilação , Prolina/química , RNA/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem , Proteínas Virais/químicaRESUMO
The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of â¼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (â¼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference-the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival.
Assuntos
Proteínas/metabolismo , Animais , Baculoviridae/genética , Pareamento Incorreto de Bases , Biopolímeros/metabolismo , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Humanos , Proteínas/genética , RNA/metabolismo , Spodoptera/citologiaRESUMO
The accumulation of base substitutions (mutations) not subject to natural selection is the neutral mutation rate. Because this rate reflects the in vivo processes involved in maintaining the integrity of genetic information, the factors that affect the neutral mutation rate are of considerable interest. Mammals exhibit two dramatically different neutral mutation rates: the CpG mutation rate, wherein the C of most CpGs (i.e., methyl-CpG) mutate at 10-50 times that of C in any other context or of any other base. The latter mutations constitute the non-CpG rate. The high CpG rate results from the spontaneous deamination of methyl-C to T and incomplete restoration of the ensuing T:G mismatches to C:Gs. Here, we determined the neutral non-CpG mutation rate as a function of CpG content by comparing sequence divergence of thousands of pairs of neutrally evolving chimpanzee and human orthologs that differ primarily in CpG content. Both the mutation rate and the mutational spectrum (transition/transversion ratio) of non-CpG residues change in parallel as sigmoidal (logistic) functions of CpG content. As different mechanisms generate transitions and transversions, these results indicate that both mutation rate and mutational processes are contingent on the local CpG content. We consider several possible mechanisms that might explain how CpG exerts these effects.
Assuntos
Composição de Bases/fisiologia , Ilhas de CpG/fisiologia , Mutação , Animais , Sequência de Bases , Ilhas de CpG/genética , DNA/genética , Análise Mutacional de DNA , Especiação Genética , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Mamíferos/genética , Dados de Sequência Molecular , Mutação/fisiologia , Pan troglodytes/genéticaRESUMO
L1 (LINE1) non-LTR retrotransposons are ubiquitous genomic parasites and the dominant transposable element in humans having generated about 40% of their genomic DNA during their ~ 100 million years (Myr) of activity in primates. L1 replicates in germ line cells and early embryos, causing genetic diversity and defects, but can be active in some somatic stem cells, tumors and during aging. L1 encodes two proteins essential for retrotransposition: ORF2p, a reverse transcriptase that contains an endonuclease domain, and ORF1p, a coiled coil mediated homo trimer, which functions as a nucleic acid chaperone. Both proteins contain highly conserved domains and preferentially bind their encoding transcript to form an L1 ribonucleoprotein (RNP), which mediates retrotransposition. However, the coiled coil has periodically undergone episodes of substantial amino acid replacement to the extent that a given L1 family can concurrently express multiple ORF1s that differ in the sequence of their coiled coils. Here we show that such distinct ORF1p sequences can become entangled forming heterotrimers when co-expressed from separate vectors and speculate on how coiled coil entanglement could affect coiled coil evolution.
RESUMO
Abundant APOBEC3 (A3) deaminase-mediated mutations can dominate the mutational landscape ('mutator phenotype') of some cancers, however, the basis of this sporadic vulnerability is unknown. We show here that elevated expression of the bifunctional DNA glycosylase, NEIL2, sensitizes breast cancer cells to A3B-mediated mutations and double-strand breaks (DSBs) by perturbing canonical base excision repair (BER). NEIL2 usurps the canonical lyase, APE1, at abasic sites in a purified BER system, rendering them poor substrates for polymerase ß. However, the nicked NEIL2 product can serve as an entry site for Exo1 in vitro to generate single-stranded DNA, which would be susceptible to both A3B and DSBs. As NEIL2 or Exo1 depletion mitigates the DNA damage caused by A3B expression, we suggest that aberrant NEIL2 expression can explain certain instances of A3B-mediated mutations.
Assuntos
Desaminases APOBEC/genética , Neoplasias da Mama/metabolismo , Citidina Desaminase/metabolismo , Reparo do DNA , Antígenos de Histocompatibilidade Menor/genética , Mutação , Desaminases APOBEC/metabolismo , Linhagem Celular Tumoral , Citidina Desaminase/genética , Humanos , Antígenos de Histocompatibilidade Menor/metabolismo , Transcrição GênicaRESUMO
Retrotransposable element insertions are 20 times more numerous per unit length of DNA in the large human genome compared to the small Drosophila genome. Whereas all Drosophila elements are subject to constant turnover (recent insertion and elimination by selection), this has not generally been the case for human retrotransposons. We suggest that a difference in recombination adopted by these organisms in response to the deleterious effects of interspersed repeated DNA can explain in part this fundamental difference between the evolutionary dynamics of fruit fly and human retrotransposons.
Assuntos
Drosophila/genética , Genoma Humano , Retroelementos , Animais , Evolução Molecular , Genoma , Humanos , Elementos Nucleotídeos Longos e Dispersos , Filogenia , Seleção Genética , Especificidade da EspécieRESUMO
L1 retrotransposons replicate (amplify) by copying (reverse transcribing) their RNA transcript into genomic DNA. The evolutionary history of L1 in mammals has been unique. In mice and humans approximately 80 million years of L1 evolution and replication produced a single evolutionary lineage of L1 elements while generating approximately 20% of the genomic mass in each species. By contrast, zebrafish contain >30 distinct L1 lineages that have generated approximately one-tenth as much DNA. We contend that, by becoming far more permissive of interspersed repeated DNA than other organisms, mammals are conducive to competition between L1 families for replicative dominance, and that this competition, perhaps for the host factors required for L1 replication, results in a single L1 lineage.
Assuntos
Peixes/genética , Elementos Nucleotídeos Longos e Dispersos , Mamíferos/genética , Animais , Evolução Biológica , Variação Genética , Humanos , FilogeniaRESUMO
L1 non-LTR retrotransposons are autonomously replicating genetic elements that profoundly affected their mammalian hosts having generated upwards of 40% or more of their genomes. Although deleterious, they remain active in most mammalian species, and thus the nature and consequences of the interaction between L1 and its host remain major issues for mammalian biology. We recently showed that L1 activity requires phosphorylation of one of its 2 encoded proteins, ORF1p, a nucleic acid chaperone and the major component of the L1RNP retrotransposition intermediate. Reversible protein phosphorylation, which is effected by interacting cascades of protein kinases, phosphatases, and ancillary proteins, is a mainstay in the regulation and coordination of many basic biological processes. Therefore, demonstrating phosphorylation-dependence of L1 activity substantially enlarged our knowledge of the scope of L1 / host interaction. However, developing a mechanistic understanding of what this means for L1 or its host is a formidable challenge, which we discuss here.
RESUMO
Species survival depends on the faithful replication of genetic information, which is continually monitored and maintained by DNA repair pathways that correct replication errors and the thousands of lesions that arise daily from the inherent chemical lability of DNA and the effects of genotoxic agents. Nonetheless, neutrally evolving DNA (not under purifying selection) accumulates base substitutions with time (the neutral mutation rate). Thus, repair processes are not 100% efficient. The neutral mutation rate varies both between and within chromosomes. For example it is 10-50 fold higher at CpGs than at non-CpG positions. Interestingly, the neutral mutation rate at non-CpG sites is positively correlated with CpG content. Although the basis of this correlation was not immediately apparent, some bioinformatic results were consistent with the induction of non-CpG mutations by DNA repair at flanking CpG sites. Recent studies with a model system showed that in vivo repair of preformed lesions (mismatches, abasic sites, single stranded nicks) can in fact induce mutations in flanking DNA. Mismatch repair (MMR) is an essential component for repair-induced mutations, which can occur as distant as 5 kb from the introduced lesions. Most, but not all, mutations involved the C of TpCpN (G of NpGpA) which is the target sequence of the C-preferring single-stranded DNA specific APOBEC deaminases. APOBEC-mediated mutations are not limited to our model system: Recent studies by others showed that some tumors harbor mutations with the same signature, as can intermediates in RNA-guided endonuclease-mediated genome editing. APOBEC deaminases participate in normal physiological functions such as generating mutations that inactivate viruses or endogenous retrotransposons, or that enhance immunoglobulin diversity in B cells. The recruitment of normally physiological error-prone processes during DNA repair would have important implications for disease, aging and evolution. This perspective briefly reviews both the bioinformatic and biochemical literature relevant to repair-induced mutagenesis and discusses future directions required to understand the mechanistic basis of this process.
Assuntos
Pareamento Incorreto de Bases , Citidina Desaminase/genética , Reparo de Erro de Pareamento de DNA , DNA de Cadeia Simples/química , Mutação , Desaminase APOBEC-1 , Sequência de Bases , Ilhas de CpG , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/metabolismo , Expressão Gênica , Genoma Humano , Humanos , Dados de Sequência Molecular , MutagêneseRESUMO
'Normal' genomic DNA contains hundreds of mismatches that are generated daily by the spontaneous deamination of C (U/G) and methyl-C (T/G). Thus, a mutagenic effect of their repair could constitute a serious genetic burden. We show here that while mismatches introduced into human cells on an SV40-based episome were invariably repaired, this process induced mutations in flanking DNA at a significantly higher rate than no mismatch controls. Most mutations involved the C of TpC, the substrate of some single strand-specific APOBEC cytidine deaminases, similar to the mutations that can typify the 'mutator phenotype' of numerous tumors. siRNA knockdowns and chromatin immunoprecipitation showed that TpC preferring APOBECs mediate the mutagenesis, and siRNA knockdowns showed that both the base excision and mismatch repair pathways are involved. That naturally occurring mispairs can be converted to mutators, represents an heretofore unsuspected source of genetic changes that could underlie disease, aging, and evolutionary change.DOI: http://dx.doi.org/10.7554/eLife.02001.001.
Assuntos
Pareamento Incorreto de Bases , Reparo do DNA , DNA/genética , Mutação , Imunoprecipitação da Cromatina , Citidina Desaminase , DNA/química , Humanos , Antígenos de Histocompatibilidade Menor , RNA Interferente Pequeno/genética , Vírus 40 dos Símios/genéticaRESUMO
The neutral mutation rate is equal to the base substitution rate when the latter is not affected by natural selection. Differences between these rates may reveal that factors such as natural selection, linkage, or a mutator locus are affecting a given sequence. We examined the neutral base substitution rate by measuring the sequence divergence of approximately 30,000 pairs of inactive orthologous L1 retrotransposon sequences interspersed throughout the human and chimpanzee genomes. In contrast to other studies, we related ortholog divergence to the time (age) that the L1 sequences resided in the genome prior to the chimpanzee and human speciation. As expected, the younger orthologs contained more hypermutable CpGs than the older ones because of their conversion to TpGs (and CpAs). Consequently, the younger orthologs accumulated more CpG mutations than the older ones during the approximately 5 million years since the human and chimpanzee lineages separated. But during this same time, the younger orthologs also accumulated more non-CpG mutations than the older ones. In fact, non-CpG and CpG mutations showed an almost perfect (R2 = 0.98) correlation for approximately 97% of the ortholog pairs. The correlation is independent of G + C content, recombination rate, and chromosomal location. Therefore, it likely reflects an intrinsic effect of CpGs, or mutations thereof, on non-CpG DNA rather than the joint manifestation of the chromosomal environment. The CpG effect is not uniform for all regions of non-CpG DNA. Therefore, the mutation rate of non-CpG DNA is contingent to varying extents on local CpG content. Aside from their implications for mutational mechanisms, these results indicate that a precise determination of a uniform genome-wide neutral mutation rate may not be attainable.
Assuntos
Ilhas de CpG , Mutação , Animais , DNA/química , Elementos de DNA Transponíveis , Feminino , Genoma , Humanos , Masculino , Mamíferos , Modelos Genéticos , Análise de Sequência de DNARESUMO
The self-replicating LINE-1 (L1) retrotransposon family is the dominant retrotransposon family in mammals and has generated 30-40% of their genomes. Active L1 families are present in modern mammals but the important question of whether these currently active families affect the genetic fitness of their hosts has not been addressed. This issue is of particular relevance to humans as Homo sapiens contains the active L1 Ta1 subfamily of the human specific Ta (L1Pa1) L1 family. Although DNA insertions generated by the Ta1 subfamily can cause genetic defects in current humans, these are relatively rare, and it is not known whether Ta1-generated inserts or any other property of Ta1 elements have been sufficiently deleterious to reduce the fitness of humans. Here we show that full-length (FL) Ta1 elements, but not the truncated Ta1 elements or SINE (Alu) insertions generated by Ta1 activity, were subject to negative selection. Thus, one or more properties unique to FL L1 elements constitute a genetic burden for modern humans. We also found that the FL Ta1 elements became more deleterious as the expansion of Ta1 has proceeded. Because this expansion is ongoing, the Ta1 subfamily almost certainly continues to decrease the fitness of modern humans.
Assuntos
Adaptação Fisiológica/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Genoma Humano/genética , Humanos , Modelos Genéticos , Polimorfismo Genético , Seleção Genética , Elementos Nucleotídeos Curtos e Dispersos/genéticaRESUMO
LINE-1 (L1) elements constitute the major family of retrotransposons in mammalian genomes. Here we report the first investigation of L1 evolution in New World monkeys (NWM). Two regions of the second open-reading frame were analyzed by two methods in three NWM species, the squirrel monkey (Saimiri sciureus), the tamarin (Saguinus oedipus), and the spider monkey (Ateles paniscus). Since these three species diverged, L1 has amplified in the Saimiri and Saguinus lineages but L1 activity seems to have been strongly reduced in the Ateles lineage. In addition, the active L1 lineage has evolved rapidly in Saimiri and Saguinus, generating species-specific subfamilies. In contrast, we found no evidence for a species-specific subfamily in Ateles, a result consistent with the low L1 activity in this species for the last approximately 25 My.
Assuntos
Cebidae/genética , Evolução Molecular , Elementos Nucleotídeos Longos e Dispersos/genética , Filogenia , Animais , Sequência de Bases , Southern Blotting , Análise por Conglomerados , Primers do DNA , Modelos Genéticos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
As humans contain a currently active L1 (LINE-1) non-LTR retrotransposon family (Ta-1), the human genome database likely provides only a partial picture of Ta-1-generated diversity. We used a non-biased method to clone Ta-1 retrotransposon-containing loci from representatives of four ethnic populations. We obtained 277 distinct Ta-1 loci and identified an additional 67 loci in the human genome database. This collection represents approximately 90% of the Ta-1 population in the individuals examined and is thus more representative of the insertional history of Ta-1 than the human genome database, which lacked approximately 40% of our cloned Ta-1 elements. As both polymorphic and fixed Ta-1 elements are as abundant in the GC-poor genomic regions as in ancestral L1 elements, the enrichment of L1 elements in GC-poor areas is likely due to insertional bias rather than selection. Although the chromosomal distribution of Ta-1 inserts is generally a function of chromosomal length and gene density, chromosome 4 significantly deviates from this pattern and has been much more hospitable to Ta-1 insertions than any other chromosome. Also, the intra-chromosomal distribution of Ta-1 elements is not uniform. Ta-1 elements tend to cluster, and the maximal gaps between Ta-1 inserts are larger than would be expected from a model of uniform random insertion.