The Origin and Evolution of Sex Peptide and Sex Peptide Receptor Interactions.

Post-mating responses play a vital role in successful reproduction across diverse species. In fruit flies, sex peptide binds to the sex peptide receptor, triggering a series of post-mating responses. However, the origin of sex peptide receptor predates the emergence of sex peptide. The evolutionary origins of the interactions between sex peptide and sex peptide receptor and the mechanisms by which they interact remain enigmatic. In this study, we used ancestral sequence reconstruction, AlphaFold2 predictions, and molecular dynamics simulations to study sex peptide-sex peptide receptor interactions and their origination. Using AlphaFold2 and long-time molecular dynamics simulations, we predicted the structure and dynamics of sex peptide-sex peptide receptor interactions. We show that sex peptide potentially binds to the ancestral states of Diptera sex peptide receptor. Notably, we found that only a few amino acid changes in sex peptide receptor are sufficient for the formation of sex peptide-sex peptide receptor interactions. Ancestral sequence reconstruction and molecular dynamics simulations further reveal that sex peptide receptor interacts with sex peptide through residues that are mostly involved in the interaction interface of an ancestral ligand, myoinhibitory peptides. We propose a potential mechanism whereby sex peptide-sex peptide receptor interactions arise from the preexisting myoinhibitory peptides-sex peptide receptor interface as well as early chance events both inside and outside the preexisting interface that created novel sex peptide-specific sex peptide-sex peptide receptor interactions. Our findings provide new insights into the origin and evolution of sex peptide-sex peptide receptor interactions and their relationship with myoinhibitory peptides-sex peptide receptor interactions.

Intermolecular Interactions Drive Protein Adaptive and Coadaptive Evolution at Both Species and Population Levels.

Proteins are the building blocks for almost all the functions in cells. Understanding the molecular evolution of proteins and the forces that shape protein evolution is essential in understanding the basis of function and evolution. Previous studies have shown that adaptation frequently occurs at the protein surface, such as in genes involved in host-pathogen interactions. However, it remains unclear whether adaptive sites are distributed randomly or at regions associated with particular structural or functional characteristics across the genome, since many proteins lack structural or functional annotations. Here, we seek to tackle this question by combining large-scale bioinformatic prediction, structural analysis, phylogenetic inference, and population genomic analysis of Drosophila protein-coding genes. We found that protein sequence adaptation is more relevant to function-related rather than structure-related properties. Interestingly, intermolecular interactions contribute significantly to protein adaptation. We further showed that intermolecular interactions, such as physical interactions, may play a role in the coadaptation of fast-adaptive proteins. We found that strongly differentiated amino acids across geographic regions in protein-coding genes are mostly adaptive, which may contribute to the long-term adaptive evolution. This strongly indicates that a number of adaptive sites tend to be repeatedly mutated and selected throughout evolution in the past, present, and maybe future. Our results highlight the important roles of intermolecular interactions and coadaptation in the adaptive evolution of proteins both at the species and population levels.

Graphene-based Pancharatnam-Berry phase metasurface in the terahertz domain for dynamically independent amplitude and phase manipulation.

Dynamic and independent amplitude and phase manipulation are the paramount demand for many advanced wavefronts engineering applications. Currently, the coupling issue between the amplitude and phase hinders the efficient modulation wavefront's further implementation. This paper proposes and numerically demonstrates the bi-layer stacked graphene Pancharatnam-Berry (P-B) phase metasurface and mono-layer graphene P-B phase metasurface to address the above problem. The simulation results show that the proposed models can achieve the independent control amplitude and phase and significantly reduce their coupling strength. Our findings offer a flexible and straightforward method for precise wave reconstruction applications such as holography, optical tweezers, and high-resolution imaging.

Fish Rhbdd3 positively regulates IFN response through RIG-I signaling pathway.

Rhomboid domain-containing protein 3 (Rhbdd3) is a member of the rhomboid family, which can modulate the innate immune response in mammals. Nonetheless, the function and regulatory mechanism of fish Rhbdd3 during viral infection have not been characterized. In this study, Rhbdd3 was firstly cloned from common carp (Cyprinus carpio) and nominated as CcRhbdd3. Phylogenetically characterization showed that CcRhbdd3 shared a relatively long evolutionary distance with its mammalian homologs. In vivo experiment demonstrated that spring viraemia of carp virus (SVCV) infection promoted the expression of CcRhbdd3 in the liver, spleen, kidney and muscle tissues. Furthermore, overexpression of CcRhbdd3 significantly inhibited SVCV propagation, whereas knockdown of CcRhbdd3 markedly promoted SVCV replication in susceptible cells. RNA-seq and following validation showed that CcRhbdd3 overexpression upregulated the expression of several RIG-I signaling related genes, including TRIM25, TRAF2, MDA5, LGP2, IFN1, IFN3, RIG-I, IRF3 and ISG15. Moreover, CcRhbdd3 promoted the expression of NF-κB, a central immune regulator. Subcellular localization experiments showed that CcRhbdd3 was primarily distributed in the cytoplasm and co-localized with Rab5 in the early endosomes. Truncation experiments further demonstrated that the C-terminus containing the ubiquitin-binding associated domain, was crucial for both the subcellular localization and antiviral activity of CcRhbdd3. The findings in this study provide new insight into the host antiviral mechanism against aquatic RNA virus infection, and will facilitate the development of therapeutic strategies for the infection of SVCV.

Behavioral and Genomic Sensory Adaptations Underlying the Pest Activity of Drosophila suzukii.

Studying how novel phenotypes originate and evolve is fundamental to the field of evolutionary biology as it allows us to understand how organismal diversity is generated and maintained. However, determining the basis of novel phenotypes is challenging as it involves orchestrated changes at multiple biological levels. Here, we aim to overcome this challenge by using a comparative species framework combining behavioral, gene expression, and genomic analyses to understand the evolutionary novel egg-laying substrate-choice behavior of the invasive pest species Drosophila suzukii. First, we used egg-laying behavioral assays to understand the evolution of ripe fruit oviposition preference in D. suzukii compared with closely related species D. subpulchrella and D. biarmipes as well as D. melanogaster. We show that D. subpulchrella and D. biarmipes lay eggs on both ripe and rotten fruits, suggesting that the transition to ripe fruit preference was gradual. Second, using two-choice oviposition assays, we studied how D. suzukii, D. subpulchrella, D. biarmipes, and D. melanogaster differentially process key sensory cues distinguishing ripe from rotten fruit during egg-laying. We found that D. suzukii's preference for ripe fruit is in part mediated through a species-specific preference for stiff substrates. Last, we sequenced and annotated a high-quality genome for D. subpulchrella. Using comparative genomic approaches, we identified candidate genes involved in D. suzukii's ability to seek out and target ripe fruits. Our results provide detail to the stepwise evolution of pest activity in D. suzukii, indicating important cues used by this species when finding a host, and the molecular mechanisms potentially underlying their adaptation to a new ecological niche.

Terahertz quasi non-diffraction Bessel vortex beam generation using three lattice types reflective metasurface.

Bandwidth, orbital-angular momentum (OAM) divergence, and mode purity are the three critical issues for the practical terahertz orbital angular momentum manipulation, especially in the next sixth-generation (6G) communication system. Here we propose the broadband high-order Bessel vortex beam carrying multiple OAM modes reflective metasurface in the terahertz domain. The simulation results agree with the theoretical expectation, and the diffracting divergence of OAM vortex beam characteristics has been alleviated. The research on the relationship between the varieties of lattice type and mode purity is also relatively scarce. Henceforth, a comparison study has been conducted between three lattice types, i.e., square lattice, triangular lattice, and concentric ring lattice. And corresponding results of the relationship of mode purity with those lattice types show that the concentric ring lattice has the best performance.

Three-site and five-site fixed-charge water models compatible with AMOEBA force field.

In a typical biomolecular simulation using Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field, the vast majority molecules in the simulation box consist of water, and these water molecules consume the most CPU power due to the explicit mutual induction effect. To improve the computational efficiency, we here develop two new nonpolarizable water models (with flexible bonds and fixed charges) that are compatible with AMOEBA solute: the 3-site AW3C and 5-site AW5C. To derive the force-field parameters for AW3C and AW5C, we fit to six experimental liquid thermodynamic properties: liquid density, enthalpy of vaporization, dielectric constant, isobaric heat capacity, isothermal compressibility and thermal expansion coefficient, at a broad range of temperatures from 261.15 to 353.15 K under 1.0 atm pressure. We further validate our AW3C and AW5C water models by showing that they can well reproduce the radial distribution function g(r), self-diffusion constant D, and hydration free energy from the AMOEBA03 water model and the experimental observations. Furthermore, we show that our AW3C and AW5C water models can greatly accelerate (>5 times) the bulk water as well as biomolecular simulations when compared to AMOEBA water. Specifically, we demonstrate that the applications of AW3C and AW5C water models to simulate a DNA duplex lead to a threefold acceleration, and in the meanwhile well maintain the structural properties as the fully polarizable AMOEBA water. We expect that our AW3C and AW5C water models hold great promise to be widely applied to simulate complex bio-molecules using the AMOEBA force field.

The facile and visualizable identification of broad-spectrum inhibitors of MDM2/p53 using co-expressed protein complexes.

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a ß-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.

SAXS-Oriented Ensemble Refinement of Flexible Biomolecules.

The conformational flexibility of a biomolecule may play a crucial role in its biological function. Small-angle x-ray scattering (SAXS) is a very popular technique for characterizing biomolecule flexibility. It can be used to determine a possible structural ensemble of the biomolecule in solution with the aid of a computer simulation. In this article, we present a tool written in Python, which iteratively runs multiple independent enhanced sampling simulations such as amplified collective motions and accelerated molecular dynamics, and an ensemble optimization method to drive the biomolecule toward an ensemble that fits the SAXS data well. The tool has been validated with a protein and an RNA system, i.e., the tandem WW domains of formin-binding protein 21 and the aptamer domain of SAM-1 riboswitch, respectively. These Python scripts are user-friendly and can be easily modified if a different simulation engine is preferred.

Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate.

Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N(1) methylation of guanine at Position 9 (m(1)G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.

Characterization of protein flexibility using small-angle x-ray scattering and amplified collective motion simulations.

Large-scale flexibility within a multidomain protein often plays an important role in its biological function. Despite its inherent low resolution, small-angle x-ray scattering (SAXS) is well suited to investigate protein flexibility and determine, with the help of computational modeling, what kinds of protein conformations would coexist in solution. In this article, we develop a tool that combines SAXS data with a previously developed sampling technique called amplified collective motions (ACM) to elucidate structures of highly dynamic multidomain proteins in solution. We demonstrate the use of this tool in two proteins, bacteriophage T4 lysozyme and tandem WW domains of the formin-binding protein 21. The ACM simulations can sample the conformational space of proteins much more extensively than standard molecular dynamics (MD) simulations. Therefore, conformations generated by ACM are significantly better at reproducing the SAXS data than are those from MD simulations.

Structural investigation of the interaction between the tandem SH3 domains of c-Cbl-associated protein and vinculin.

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.

The origin and structural evolution of de novo genes in Drosophila.

Recent studies reveal that de novo gene origination from previously non-genic sequences is a common mechanism for gene innovation. These young genes provide an opportunity to study the structural and functional origins of proteins. Here, we combine high-quality base-level whole-genome alignments and computational structural modeling to study the origination, evolution, and protein structures of lineage-specific de novo genes. We identify 555 de novo gene candidates in D. melanogaster that originated within the Drosophilinae lineage. Sequence composition, evolutionary rates, and expression patterns indicate possible gradual functional or adaptive shifts with their gene ages. Surprisingly, we find little overall protein structural changes in candidates from the Drosophilinae lineage. We identify several candidates with potentially well-folded protein structures. Ancestral sequence reconstruction analysis reveals that most potentially well-folded candidates are often born well-folded. Single-cell RNA-seq analysis in testis shows that although most de novo gene candidates are enriched in spermatocytes, several young candidates are biased towards the early spermatogenesis stage, indicating potentially important but less emphasized roles of early germline cells in the de novo gene origination in testis. This study provides a systematic overview of the origin, evolution, and protein structural changes of Drosophilinae-specific de novo genes.

The Origin and Evolution of Sex Peptide and Sex Peptide Receptor Interactions.

Post-mating responses play a vital role in successful reproduction across diverse species. In fruit flies, sex peptide (SP) binds to the sex peptide receptor (SPR), triggering a series of post-mating responses. However, the origin of SPR predates the emergence of SP. The evolutionary origins of the interactions between SP and SPR and the mechanisms by which they interact remain enigmatic. In this study, we used ancestral sequence reconstruction, AlphaFold2 predictions, and molecular dynamics simulations to study SP-SPR interactions and their origination. Using AlphaFold2 and long-time molecular dynamics (MD) simulations, we predicted the structure and dynamics of SP-SPR interactions. We show that SP potentially binds to the ancestral states of Diptera SPR. Notably, we found that only a few amino acid changes in SPR are sufficient for the formation of SP-SPR interactions. Ancestral sequence reconstruction and MD simulations further reveal that SPR interacts with SP through residues that are mostly involved in the interaction interface of an ancestral ligand, myoinhibitory peptides (MIPs). We propose a potential mechanism whereby SP-SPR interactions arise from the pre-existing MIP-SPR interface as well as early chance events both inside and outside the pre-existing interface that created novel SP-specific SP-SPR interactions. Our findings provide new insights into the origin and evolution of SP-SPR interactions and their relationship with MIP-SPR interactions.

Structural insights into the recognition of the A/T-rich motif in target gene promoters by the LMX1a homeobox domain.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development.

The origin and structural evolution of de novo genes in Drosophila.

Although previously thought to be unlikely, recent studies have shown that de novo gene origination from previously non-genic sequences is a relatively common mechanism for gene innovation in many species and taxa. These young genes provide a unique set of candidates to study the structural and functional origination of proteins. However, our understanding of their protein structures and how these structures originate and evolve are still limited, due to a lack of systematic studies. Here, we combined high-quality base-level whole genome alignments, bioinformatic analysis, and computational structure modeling to study the origination, evolution, and protein structure of lineage-specific de novo genes. We identified 555 de novo gene candidates in D. melanogaster that originated within the Drosophilinae lineage. We found a gradual shift in sequence composition, evolutionary rates, and expression patterns with their gene ages, which indicates possible gradual shifts or adaptations of their functions. Surprisingly, we found little overall protein structural changes for de novo genes in the Drosophilinae lineage. Using Alphafold2, ESMFold, and molecular dynamics, we identified a number of de novo gene candidates with protein products that are potentially well-folded, many of which are more likely to contain transmembrane and signal proteins compared to other annotated protein-coding genes. Using ancestral sequence reconstruction, we found that most potentially well-folded proteins are often born folded. Interestingly, we observed one case where disordered ancestral proteins become ordered within a relatively short evolutionary time. Single-cell RNA-seq analysis in testis showed that although most de novo genes are enriched in spermatocytes, several young de novo genes are biased in the early spermatogenesis stage, indicating potentially important but less emphasized roles of early germline cells in the de novo gene origination in testis. This study provides a systematic overview of the origin, evolution, and structural changes of Drosophilinae-specific de novo genes.

Method for efficient calculating earth pressure of retaining wall considering plant transpiration.

An accurate estimation of earth pressure on retaining walls is imperative to achieving its design. This paper presents an analytical method framework that considers the effect of plant transpiration relative to the traditional calculation approaches. Specifically, a closed-form solution for one-dimensional steady unsaturated flow considering plant transpiration is incorporated into a representation of effective stress to obtain the changes in matric suction, and effective stress. The representations are used to extend Hooke's law and Rankine's earth pressure theory to determine at-rest, active, and passive earth pressures. Subsequently, the analytical method is used in a series of analysis case studies on the influence of root architecture types, transpiration rates, and soil types on earth pressure, to reveal that it can rapidly obtain the earth pressure. Notably, the effect of plant transpiration on earth pressure is significant. Furthermore, it is found that soil types and transpiration rates have a larger influence than root architecture types. Collectively, the research not only reveals the effect of plant on earth pressure for retaining wall, but also provides a theoretical basis for further exploration of the contribution of plants to the stability of retaining wall.

The two-component system expression patterns and immune regulatory mechanism of Vibrio parahaemolyticus with different genotypes at the early stage of infection in THP-1 cells.

Vibrio parahaemolyticus must endure various challenging circumstances while being swallowed by phagocytes of the innate immune system. Moreover, bacteria should recognize and react to environmental signals quickly in host cells. Two-component system (TCS) is an important way for bacteria to perceive external environmental signals and transmit them to the interior to trigger the associated regulatory mechanism. However, the regulatory function of V. parahaemolyticus TCS in innate immune cells is unclear. Here, the expression patterns of TCS in V. parahaemolyticus-infected THP-1 cell-derived macrophages at the early stage were studied for the first time. Based on protein-protein interaction network analysis, we mined and analyzed seven critical TCS genes with excellent research value in the V. parahaemolyticus regulating macrophages, as shown below. VP1503, VP1502, VPA0021, and VPA0182 could regulate the ATP-binding-cassette (ABC) transport system. VP1735, uvrY, and peuR might interact with thermostable hemolysin proteins, DNA cleavage-related proteins, and TonB-dependent siderophore enterobactin receptor, respectively, which may assist V. parahaemolyticus in infected macrophages. Subsequently, the potential immune escape pathways of V. parahaemolyticus regulating macrophages were explored by RNA-seq. The results showed that V. parahaemolyticus might infect macrophages by controlling apoptosis, actin cytoskeleton, and cytokines. In addition, we found that the TCS (peuS/R) could enhance the toxicity of V. parahaemolyticus to macrophages and might contribute to the activation of macrophage apoptosis. IMPORTANCE This study could offer crucial new insights into the pathogenicity of V. parahaemolyticus without tdh and trh genes. In addition, we also provided a novel direction of inquiry into the pathogenic mechanism of V. parahaemolyticus and suggested several TCS key genes that may assist V. parahaemolyticus in innate immune regulation and interaction.

CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.