A protein architecture guided screen for modification dependent restriction endonucleases

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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The Efficiency of 5-Methylcytosine Identification in DNA of Escherichia coli Cells that Carry Bacterial DNA Methyltransferase Genes Using an Oxford Nanopore Device

BIOPHYSICS ◽

10.1134/s0006350920060056 ◽

2020 ◽

Vol 65 (6) ◽

pp. 889-893

Author(s):

V. V. Ilinsky ◽

E. M. Kozlova ◽

S. Kh. Degtyarev ◽

N. K. Yankovsky ◽

V. J. Makeev

Keyword(s):

Escherichia Coli ◽

Dna Methyltransferase ◽

Bacterial Dna ◽

Escherichia Coli Cells ◽

Oxford Nanopore

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Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

Nucleic Acids Research ◽

10.1093/nar/gkaa938 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11495-11509

Author(s):

Michael Dukatz ◽

Sabrina Adam ◽

Mahamaya Biswal ◽

Jikui Song ◽

Pavel Bashtrykov ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Flanking Sequence ◽

Cpg Sites ◽

Binding Interface ◽

Target Sites ◽

Flanking Sequences

Abstract DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

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Origin and Evolution of DNA methyltransferases (DNMT) along the tree of life: A multi-genome survey

10.1101/2020.04.09.033167 ◽

2020 ◽

Author(s):

Madhumita Bhattacharyya ◽

Subhajyoti De ◽

Saikat Chakrabarti

Keyword(s):

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

Dna Methyltransferases ◽

Tree Of Life ◽

Sequence Length ◽

Steady Increase ◽

Human Populations ◽

Nucleotide Polymorphisms ◽

Dnmt Family

AbstractBackgroundCytosine methylation is a common DNA modification found in most eukaryotic organisms including plants, animals, and fungi. (Cytosine-5)-DNA methyltransferases (C5-DNA MTases) belong to the DNMT family of enzymes that catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to cytosine residues of DNA. In mammals, four members of the DNMT family have been reported: DNMT1, DNMT3a, DNMT3b and DNMT3L, but only DNMT1, DNMT3a and DNMT3b possess methyltransferase activity. There have been many reports about the methylation landscape in different organisms yet there is no systematic report of how the enzyme DNA (C5) methyltransferases have evolved in different organisms.ResultDNA methyltransferases are found to be present in all three domains of life. However, significant variability has been observed in length, copy number and sequence identity when compared across kingdoms. Sequence conservation is greatly increased in invertebrates and vertebrates compared to other groups. Similarly, sequence length has been found to be increased while domain lengths remain more or less conserved. Vertebrates are also found to be associated with more conserved DNMT domains. Finally, comparison between single nucleotide polymorphisms (SNPs) prevailing in human populations and evolutionary changes in DNMT vertebrate alignment revealed that most of the SNPs were conserved in vertebrates.ConclusionThe sequences (including the catalytic domain and motifs) and structure of the DNMT enzymes have been evolved greatly from bacteria to vertebrates with a steady increase in complexity and specificity. This study provides a systematic report of the evolution of DNA methyltransferase enzyme across different lineages of tree of life.

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Secretion of streptokinase fusion proteins from Escherichia coli cells through the hemolysin transporter

Gene ◽

10.1016/0378-1119(95)00395-m ◽

1995 ◽

Vol 163 (1) ◽

pp. 53-57 ◽

Cited By ~ 12

Author(s):

Izabella Kern ◽

Piotr Ceglowski

Keyword(s):

Escherichia Coli ◽

Fusion Proteins ◽

Escherichia Coli Cells

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Faculty Opinions recommendation of RNA dynamics in live Escherichia coli cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1021598.246622 ◽

2004 ◽

Author(s):

Tina Henkin

Keyword(s):

Escherichia Coli ◽

Rna Dynamics ◽

Escherichia Coli Cells

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Faculty Opinions recommendation of Deletion of penicillin-binding protein 5 (PBP5) sensitises Escherichia coli cells to beta-lactam agents.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1649962.1152060 ◽

2010 ◽

Author(s):

Samuel Kariuki

Keyword(s):

Escherichia Coli ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Beta Lactam ◽

Escherichia Coli Cells

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Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

Recent Patents on Biotechnology ◽

10.2174/1872208314999200730115018 ◽

2020 ◽

Vol 14 (4) ◽

pp. 269-282

Author(s):

Sadra S. Tehrani ◽

Golnaz Goodarzi ◽

Mohsen Naghizadeh ◽

Seyyed H. Khatami ◽

Ahmad Movahedpour ◽

...

Keyword(s):

Escherichia Coli ◽

Signal Peptide ◽

Fusion Proteins ◽

Inclusion Bodies ◽

Clinical Aspects ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

E Coli ◽

Secretory Production ◽

Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

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