Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease

Abstract Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

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Degenerate Sequence Recognition by AdpA

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409785x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C214-C214

Author(s):

Jun Ohtsuka ◽

Ming Dong Yao ◽

Koji Nagata ◽

Ken-ichi Miyazono ◽

Yuehua Zhi ◽

...

Keyword(s):

Crystal Structure ◽

Consensus Sequence ◽

Streptomyces Griseus ◽

Morphological Differentiation ◽

Aerial Mycelium ◽

Sequence Specificity ◽

Mobility Shift ◽

Sequence Recognition ◽

Regulatory Cascade ◽

Arginine Residues

AdpA is the central transcriptional factor in the A-factor regulatory cascade of Streptomyces griseus and activates hundreds of genes required for both secondary metabolism and morphological differentiation, leading to onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. It has been shown that AdpA binds to over 500 operator regions with the loosely conserved consensus sequence, 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; and N: any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To reveal the molecular basis of the low nucleotide sequence specificity, we have determined the crystal structure of the complex of DNA-binding domain of AdpA and a 14-mer duplex DNA with two-nucleotide overhangs at 5'-ends at 2.95-Å resolution. The crystal structure and electrophoretic mobility-shift assays showed that only two arginine residues, Arg262 and Arg266, are involved in the sequence recognition and determine the nucleotide specificity/preference of continuous five base-pairs of positions 1–5 in the consensus sequence. These results partially explain how AdpA directly controls hundreds of genes.

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The ‘Tommy Atkins’ mango genome reveals candidate genes for fruit quality

BMC Plant Biology ◽

10.1186/s12870-021-02858-1 ◽

2021 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Ian S. E. Bally ◽

◽

Aureliano Bombarely ◽

Alan H. Chambers ◽

Yuval Cohen ◽

...

Keyword(s):

Candidate Genes ◽

Fruit Quality ◽

De Novo ◽

Mapping Population ◽

Mangifera Indica ◽

Consensus Sequence ◽

Fruit Size ◽

Hybrid Population ◽

High Molecular Weight Dna ◽

Tropical Fruit

Abstract Background Mango, Mangifera indica L., an important tropical fruit crop, is grown for its sweet and aromatic fruits. Past improvement of this species has predominantly relied on chance seedlings derived from over 1000 cultivars in the Indian sub-continent with a large variation for fruit size, yield, biotic and abiotic stress resistance, and fruit quality among other traits. Historically, mango has been an orphan crop with very limited molecular information. Only recently have molecular and genomics-based analyses enabled the creation of linkage maps, transcriptomes, and diversity analysis of large collections. Additionally, the combined analysis of genomic and phenotypic information is poised to improve mango breeding efficiency. Results This study sequenced, de novo assembled, analyzed, and annotated the genome of the monoembryonic mango cultivar ‘Tommy Atkins’. The draft genome sequence was generated using NRGene de-novo Magic on high molecular weight DNA of ‘Tommy Atkins’, supplemented by 10X Genomics long read sequencing to improve the initial assembly. A hybrid population between ‘Tommy Atkins’ x ‘Kensington Pride’ was used to generate phased haplotype chromosomes and a highly resolved phased SNP map. The final ‘Tommy Atkins’ genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango and included ~ 86% of the ~ 439 Mb haploid mango genome. Skim sequencing identified ~ 3.3 M SNPs using the ‘Tommy Atkins’ x ‘Kensington Pride’ mapping population. Repeat masking identified 26,616 genes with a median length of 3348 bp. A whole genome duplication analysis revealed an ancestral 65 MYA polyploidization event shared with Anacardium occidentale. Two regions, one on LG4 and one on LG7 containing 28 candidate genes, were associated with the commercially important fruit size characteristic in the mapping population. Conclusions The availability of the complete ‘Tommy Atkins’ mango genome will aid global initiatives to study mango genetics.

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Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽

10.3390/molecules26133986 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3986

Author(s):

Cécilia Hognon ◽

Antonio Monari

Keyword(s):

Dna Cleavage ◽

Catalytic Mechanism ◽

Dna Lesions ◽

Cleavage Reaction ◽

Dynamic Simulations ◽

Important Target ◽

System Adaptation ◽

Dna Strands ◽

Dna Strand ◽

Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

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Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages

Blood ◽

10.1182/blood-2005-01-0080 ◽

2005 ◽

Vol 106 (6) ◽

pp. 1938-1947 ◽

Cited By ~ 96

Author(s):

Tomohiko Tamura ◽

Pratima Thotakura ◽

Tetsuya S. Tanaka ◽

Minoru S. H. Ko ◽

Keiko Ozato

Keyword(s):

Transcription Factor ◽

Cystatin C ◽

Target Genes ◽

De Novo ◽

Consensus Sequence ◽

Binding Motif ◽

Microarray Gene Expression ◽

Cathepsin C ◽

Cis Element

Abstract Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

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Spaced Seed Data Structures forDe NovoAssembly

International Journal of Genomics ◽

10.1155/2015/196591 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Inanç Birol ◽

Justin Chu ◽

Hamid Mohamadi ◽

Shaun D. Jackman ◽

Karthika Raghavan ◽

...

Keyword(s):

Data Structure ◽

Data Structures ◽

De Novo ◽

Bloom Filters ◽

De Bruijn Graph ◽

Sequence Specificity ◽

Sequencing Errors ◽

Spaced Seeds ◽

Read Error Correction ◽

Seed Data

De novoassembly of the genome of a species is essential in the absence of a reference genome sequence. Many scalable assembly algorithms use the de Bruijn graph (DBG) paradigm to reconstruct genomes, where a table of subsequences of a certain length is derived from the reads, and their overlaps are analyzed to assemble sequences. Despite longer subsequences unlocking longer genomic features for assembly, associated increase in compute resources limits the practicability of DBG over other assembly archetypes already designed for longer reads. Here, we revisit the DBG paradigm to adapt it to the changing sequencing technology landscape and introduce three data structure designs for spaced seeds in the form of paired subsequences. These data structures address memory and run time constraints imposed by longer reads. We observe that when a fixed distance separates seed pairs, it provides increased sequence specificity with increased gap length. Further, we note that Bloom filters would be suitable to implicitly store spaced seeds and be tolerant to sequencing errors. Building on this concept, we describe a data structure for tracking the frequencies of observed spaced seeds. These data structure designs will have applications in genome, transcriptome and metagenome assemblies, and read error correction.

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A QM/MM Study of Acylphosphatase Reveals the Nucleophilic-Attack and Ensuing Carbonyl-Assisted Catalytic Mechanisms

10.26434/chemrxiv.11984448.v1 ◽

2020 ◽

Author(s):

zheng zhao ◽

Phil bourne ◽

Hao Hu ◽

Huanyu Chu

Keyword(s):

Energy Barrier ◽

Potential Energy Surfaces ◽

Catalytic Mechanism ◽

Interaction Networks ◽

Nucleophilic Attack ◽

Reaction Coordinates ◽

Transition State Stabilization ◽

Catalytic Mechanisms ◽

Energy Surfaces

Acylphosphatase is one of the vital enzymes in many organs/tissues to catalyze an acylphosphate molecule into carboxylate and phosphate. Here we use a combined <i>ab initio</i> QM/MM approach to reveal the catalytic mechanism of the benzoylphosphate-bound acylphosphatase system. Using a multi-dimensional reaction-coordinates-driving scheme, we obtained a detailed catalytic process including one nucleophilic-attack and then an ensuing carbonyl-shuttle catalytic mechanism by calculating two-dimensional potential energy surfaces. We also obtained an experiment-agreeable energy barrier and validated the role of the key amino acid Asn38. Additionally, we qualified the transition state stabilization strategy based on the amino acids-contributed interaction networks revealed in the enzymatic environment. This study provided usefule insights into the underlying catalytic mechanism to contribute to disease-involved research.

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A haplotype-resolved, de novo genome assembly for the wood tiger moth (Arctia plantaginis) through trio binning

GigaScience ◽

10.1093/gigascience/giaa088 ◽

2020 ◽

Vol 9 (8) ◽

Cited By ~ 2

Author(s):

Eugenie C Yen ◽

Shane A McCarthy ◽

Juan A Galarza ◽

Tomas N Generalovic ◽

Sarah Pelan ◽

...

Keyword(s):

Population Structure ◽

Genome Assembly ◽

De Novo ◽

Consensus Sequence ◽

Parental Origin ◽

De Novo Genome Assembly ◽

Geographic Population ◽

Long Reads ◽

Tiger Moth ◽

Innovative Solution

ABSTRACT Background Diploid genome assembly is typically impeded by heterozygosity because it introduces errors when haplotypes are collapsed into a consensus sequence. Trio binning offers an innovative solution that exploits heterozygosity for assembly. Short, parental reads are used to assign parental origin to long reads from their F1 offspring before assembly, enabling complete haplotype resolution. Trio binning could therefore provide an effective strategy for assembling highly heterozygous genomes, which are traditionally problematic, such as insect genomes. This includes the wood tiger moth (Arctia plantaginis), which is an evolutionary study system for warning colour polymorphism. Findings We produced a high-quality, haplotype-resolved assembly for Arctia plantaginis through trio binning. We sequenced a same-species family (F1 heterozygosity ∼1.9%) and used parental Illumina reads to bin 99.98% of offspring Pacific Biosciences reads by parental origin, before assembling each haplotype separately and scaffolding with 10X linked reads. Both assemblies are contiguous (mean scaffold N50: 8.2 Mb) and complete (mean BUSCO completeness: 97.3%), with annotations and 31 chromosomes identified through karyotyping. We used the assembly to analyse genome-wide population structure and relationships between 40 wild resequenced individuals from 5 populations across Europe, revealing the Georgian population as the most genetically differentiated with the lowest genetic diversity. Conclusions We present the first invertebrate genome to be assembled via trio binning. This assembly is one of the highest quality genomes available for Lepidoptera, supporting trio binning as a potent strategy for assembling heterozygous genomes. Using our assembly, we provide genomic insights into the geographic population structure of A. plantaginis.

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Role of 2-Phosphoglycolate Phosphatase of Escherichia coli in Metabolism of the 2-Phosphoglycolate Formed in DNA Repair

Journal of Bacteriology ◽

10.1128/jb.185.19.5815-5821.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5815-5821 ◽

Cited By ~ 38

Author(s):

Maria Teresa Pellicer ◽

Maria Felisa Nuñez ◽

Juan Aguilar ◽

Josefa Badia ◽

Laura Baldoma

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Catalytic Mechanism ◽

Constitutive Expression ◽

Catalytic Efficiency ◽

Phosphoglycolate Phosphatase ◽

General Metabolism ◽

Functional Features ◽

Dna Strand ◽

Cellular Dna

ABSTRACT The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (k cat/Km ), which enabled the conversion of this substrate even at low intracellular concentrations. A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism. Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions. We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3′-phosphoglycolate ends. DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin. The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism. We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery.

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Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5547 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5547-5557 ◽

Cited By ~ 16

Author(s):

E Remboutsika ◽

G B Kohlhaw

Keyword(s):

Consensus Sequence ◽

Binding Motif ◽

Mutant Form ◽

Amino Acid Residues ◽

Molecular Architecture ◽

Recombinant Peptide ◽

Mutant Peptide ◽

Target Dna ◽

Normal Spacing ◽

Dna Strand

The Leu3 protein (Leu3p) of Saccharomyces cerevisiae is a pleiotropic transregulator that can function both as an activator and as a repressor of transcription. It binds to upstream promoter elements (UASLEU) with the consensus sequence 5'-GCCGGNNCCGGC-3'. The DNA-binding motif of Leu3p belongs to the family of Zn(II)2-Cys6 clusters. The motif is located between amino acid residues 37 and 67 of the 886-residue protein. In this study, we used a recombinant peptide consisting of residues 17 to 147 to explore the interaction between Leu3p and its cognate DNA. We found that the Leu3p(17-147) peptide is a monomer in the absence of UASLEU but assumes a dimeric structure when the DNA is present. Results of protein-DNA cross-linking and methylation and ethylation interference footprinting experiments show that the Leu3p(17-147) dimer interacts symmetrically with two contact triplets separated by 6 bp and suggest that the peptide approaches its target DNA in such a way that each subunit is positioned closer to one DNA strand than to the other. The binding of Leu3p is strongly affected by the spacing between the contact triplets of the UASLEU and by the type of triplet. Binding occurs when the triplets are 6 bp apart (normal spacing) but fails to occur when the triplets are 0, 5, or 8 bp apart. Weak binding occurs when the triplets are 7 bp apart. Binding does not occur when the UASLEU triplets (GCC....GGC) are replaced with triplets found in the UAS elements for Gal4p, Put3p, and Ppr1p (CGG....CCG). The apparent Kd for the normal Leu3p(17-147)-UASLEU complex is about 3 nM. A mutant form of Leu3p(17-147) in which the histidine at position 50 has been replaced with cysteine binds UASLEU with significantly greater affinity (apparent Kd of about 0.7 nM), even though the interaction between the mutant peptide and target DNA appears to be unchanged. Interestingly, repression of basal-level transcription, which is a hallmark property of the wild-type Leu3p(17-147) peptide, is largely lost with the mutant peptide, indicating that there is no direct correlation between strength of binding and repression.

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Human DNMT1 transition state structure

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522491113 ◽

2016 ◽

Vol 113 (11) ◽

pp. 2916-2921 ◽

Cited By ~ 35

Author(s):

Quan Du ◽

Zhen Wang ◽

Vern L. Schramm

Keyword(s):

Transition State ◽

Dna Methyltransferase ◽

Catalytic Mechanism ◽

Isotope Effects ◽

Nucleophilic Attack ◽

Kinetic Isotope ◽

Methyl Transfer ◽

Major Transition ◽

Methylation Patterns ◽

Rate Limiting

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys1226 to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys1226 attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.

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