Cell cycle regulated Methyltransferase

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The X-ray crystal structure Caulobacter crescentus CcrM showing the CcrM homodimer in complex with double strand DNA. The protein monomers are shown in blue and green while the DNA is shown in tan. The DNA Bases in the GANTC recognition site are shown in all atom resolution. Caulobacter crescentus CcrM.png
The X-ray crystal structure Caulobacter crescentus CcrM showing the CcrM homodimer in complex with double strand DNA. The protein monomers are shown in blue and green while the DNA is shown in tan. The DNA Bases in the GANTC recognition site are shown in all atom resolution.

CcrM (or M.CcrMI) is an orphan DNA methyltransferase, that is involved in controlling gene expression in most Alphaproteobacteria. This enzyme modifies DNA by catalyzing the transference of a methyl group from the S-adenosyl-L methionine substrate to the N6 position of an adenine base in the sequence 5'-GANTC-3' with high specificity. [1] In some lineages such as SAR11, the homologous enzymes possess 5'-GAWTC-3' specificity. [2] In Caulobacter crescentus Ccrm is produced at the end of the replication cycle when Ccrm recognition sites are hemimethylated, rapidly methylating the DNA. CcrM is essential in other Alphaproteobacteria but its role is not yet determined. CcrM is a highly specific methyltransferase with a novel DNA recognition mechanism. [3]

Contents

CcrM role in cell cycle regulation

Methylations are epigenetic modification that, in eukaryotes, regulates processes as cell differentiation, and embryogenesis, [4] while in prokaryotes can have a role in self recognition, protecting the DNA from being cleaved by the restriction endonuclease system, [5] or for gene regulation. The first function is controlled by the restriction methylation system while the second by Orphan MTases as Dam and CcrM. [6]

CcrM role have been characterized in the marine model organism Caulobacter crescentus, which is suitable for the study of cell cycle and epigenetics as it asymmetrically divides generating different progeny, a stalked and a swarmer cell, with different phenotypes and gene regulation. The swarmer cell has a single flagellum and polar pili and is characterized by its mobility, while the stacked cell has a stalk and is fixed to the substrate. The stacked cell enters immediately in S-phase, while the swarmer cell stays in G1-phase and will differentiate to a stacked cell before entering the S-phase again. [7]

The stacked cell in S phase will replicate its DNA in a semiconservative manner producing two hemimethylated DNA double strands that will be rapidly methylated by the Methyltransferase CcrM, which is only produced at the end of the S phase. The enzyme will methylate more than 4 thousand 5'-GANTC-3' sites in around 20 minutes, and then it will be degraded by the LON protease. [8] This fast methylation plays an important role in the transcriptional control of several genes and controls the cell differentiation. CcrM expression is regulated by the CtrA master regulator, and in addition various 5'-GANTC-3' sites methylation sites regulate CcrM expression, which will only occur at the end of the S phase when this sites are hemimethylated. In this process CtrA regulates the expression of CcrM and more than 1000 genes in the pre-divisional state, [9] and SciP prevents the activation of CcrM transcription in non replicative cells.

CcrM role in Alphaproteobacteria

Orphan MTases are common in bacteria and archea [10] CcrM is found in almost every group of Alphaproteobacteria , excepting in Rickettsiales and Magnetococcales, and homologs can be found in Campylobacterota and Gammaproteobacteria . [11] Alphaproteobacteria are organisms with different life stages from free living to substrate associated, some of them are intracellular pathogens of plants, animal and even human, [12] in those groups the CcrMs must have an important role in cell cycle progression. [13]

CcrM miss regulation have shown to produce severe miss control of cell cycle regulation and differentiation in various Alphaproteobacteria; C. crescentus , the plant symbiont Sinorhizobium meliloti [14] and in the human pathogen Brucella abortus. [15] Also CcrM gene has proven to be essential for the viability of various Alphaproteobacteria. [12]

Structure and DNA recognition mechanism

CcrM is a type II DNA Methyltransferase, that transfer a methyl group from the methyl donor SAM to the N6 of an adenine in a 5'-GANTC-3' recognition sites of hemimethylated DNA. Based on the order of the conserved motifs that form the SAM binding, the active site and the target recognition domain in the sequence of CcrM it can be classified as a β-class adenine N6 Methyltransferase. [16] CcrM homologs in Alphaproteobacteria have an 80 residues C terminal domain, [11] with non well characterized function. [17]

CcrM is characterized by a high degree of sequence discrimination, showing a very high specificity for GANTC sites over AANTC sites , being able to recognize and methylate this sequence in both double and single strand DNA. [1] CcrM in complex with a dsDNA structure was resolved, showing that the enzyme presents a novel DNA interaction mechanism, opening a bubble in the DNA recognition site (The concerted mechanism of Methyltransferases relies in the flip of the target base), the enzyme interacts with DNA forming an homodimer with differential monomer interactions. [3]

CcrM is a highly efficient enzyme capable of methylating a high number of 5'-GANTC-3' sites in low time, however if the enzyme is processive (the enzyme binds to the DNA and methylate several methylation sites before dissociation) or distributive (the enzyme dissociates from DNA after each methylation) it is still in discussion. First reports indicated the second case, [18] however more recent characterisation of CcrM indicate that it is a processive enzyme. [19]

Related Research Articles

<span class="mw-page-title-main">Epigenetics</span> Study of DNA modifications that do not change its sequence

In biology, epigenetics is the study of heritable traits, or a stable change of cell function, that happen without changes to the DNA sequence. The Greek prefix epi- in epigenetics implies features that are "on top of" or "in addition to" the traditional genetic mechanism of inheritance. Epigenetics usually involves a change that is not erased by cell division, and affects the regulation of gene expression. Such effects on cellular and physiological phenotypic traits may result from environmental factors, or be part of normal development. They can lead to cancer.

Methylation, in the chemical sciences, is the addition of a methyl group on a substrate, or the substitution of an atom by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These terms are commonly used in chemistry, biochemistry, soil science, and biology.

<span class="mw-page-title-main">Transcription (biology)</span> Process of copying a segment of DNA into RNA

Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins produce messenger RNA (mRNA). Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs).

<span class="mw-page-title-main">DNA methyltransferase</span> Class of enzymes

In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.

<i>Caulobacter crescentus</i> Species of bacterium

Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams. The taxon is more properly known as Caulobacter vibrioides.

<span class="mw-page-title-main">DNA methylation</span> Biological process

DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis.

Histone methylation is a process by which methyl groups are transferred to amino acids of histone proteins that make up nucleosomes, which the DNA double helix wraps around to form chromosomes. Methylation of histones can either increase or decrease transcription of genes, depending on which amino acids in the histones are methylated, and how many methyl groups are attached. Methylation events that weaken chemical attractions between histone tails and DNA increase transcription because they enable the DNA to uncoil from nucleosomes so that transcription factor proteins and RNA polymerase can access the DNA. This process is critical for the regulation of gene expression that allows different cells to express different genes.

<span class="mw-page-title-main">Methyltransferase</span> Group of methylating enzymes

Methyltransferases are a large group of enzymes that all methylate their substrates but can be split into several subclasses based on their structural features. The most common class of methyltransferases is class I, all of which contain a Rossmann fold for binding S-Adenosyl methionine (SAM). Class II methyltransferases contain a SET domain, which are exemplified by SET domain histone methyltransferases, and class III methyltransferases, which are membrane associated. Methyltransferases can also be grouped as different types utilizing different substrates in methyl transfer reactions. These types include protein methyltransferases, DNA/RNA methyltransferases, natural product methyltransferases, and non-SAM dependent methyltransferases. SAM is the classical methyl donor for methyltransferases, however, examples of other methyl donors are seen in nature. The general mechanism for methyl transfer is a SN2-like nucleophilic attack where the methionine sulfur serves as the leaving group and the methyl group attached to it acts as the electrophile that transfers the methyl group to the enzyme substrate. SAM is converted to S-Adenosyl homocysteine (SAH) during this process. The breaking of the SAM-methyl bond and the formation of the substrate-methyl bond happen nearly simultaneously. These enzymatic reactions are found in many pathways and are implicated in genetic diseases, cancer, and metabolic diseases. Another type of methyl transfer is the radical S-Adenosyl methionine (SAM) which is the methylation of unactivated carbon atoms in primary metabolites, proteins, lipids, and RNA.

<span class="mw-page-title-main">DNA adenine methylase</span> Prokaryotic enzyme

DNA adenine methylase, (Dam) (also site-specific DNA-methyltransferase (adenine-specific), EC 2.1.1.72, modification methylase, restriction-modification system) is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time. It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">DNMT1</span> Protein-coding gene in the species Homo sapiens

DNA (cytosine-5)-methyltransferase 1(Dnmt1) is an enzyme that catalyzes the transfer of methyl groups to specific CpG sites in DNA, a process called DNA methylation. In humans, it is encoded by the DNMT1 gene. Dnmt1 forms part of the family of DNA methyltransferase enzymes, which consists primarily of DNMT1, DNMT3A, and DNMT3B.

DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.

<span class="mw-page-title-main">DNA demethylation</span> Removal of a methyl group from one or more nucleotides within a DNA molecule.

For molecular biology in mammals, DNA demethylation causes replacement of 5-methylcytosine (5mC) in a DNA sequence by cytosine (C). DNA demethylation can occur by an active process at the site of a 5mC in a DNA sequence or, in replicating cells, by preventing addition of methyl groups to DNA so that the replicated DNA will largely have cytosine in the DNA sequence.

Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.

<span class="mw-page-title-main">EHMT1</span> Protein-coding gene in the species Homo sapiens

Euchromatic histone-lysine N-methyltransferase 1, also known as G9a-like protein (GLP), is a protein that in humans is encoded by the EHMT1 gene.

<i>N</i><sup>6</sup>-Methyladenosine Modification in mRNA, DNA

N6-Methyladenosine (m6A) was originally identified and partially characterised in the 1970s, and is an abundant modification in mRNA and DNA. It is found within some viruses, and most eukaryotes including mammals, insects, plants and yeast. It is also found in tRNA, rRNA, and small nuclear RNA (snRNA) as well as several long non-coding RNA, such as Xist.

<span class="mw-page-title-main">Cancer epigenetics</span> Field of study in cancer research

Cancer epigenetics is the study of epigenetic modifications to the DNA of cancer cells that do not involve a change in the nucleotide sequence, but instead involve a change in the way the genetic code is expressed. Epigenetic mechanisms are necessary to maintain normal sequences of tissue specific gene expression and are crucial for normal development. They may be just as important, if not even more important, than genetic mutations in a cell's transformation to cancer. The disturbance of epigenetic processes in cancers, can lead to a loss of expression of genes that occurs about 10 times more frequently by transcription silencing than by mutations. As Vogelstein et al. points out, in a colorectal cancer there are usually about 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. However, in colon tumors compared to adjacent normal-appearing colonic mucosa, there are about 600 to 800 heavily methylated CpG islands in the promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. Manipulation of epigenetic alterations holds great promise for cancer prevention, detection, and therapy. In different types of cancer, a variety of epigenetic mechanisms can be perturbed, such as the silencing of tumor suppressor genes and activation of oncogenes by altered CpG island methylation patterns, histone modifications, and dysregulation of DNA binding proteins. There are several medications which have epigenetic impact, that are now used in a number of these diseases.

Within the field of molecular biology, the epitranscriptome includes all the biochemical modifications of the RNA within a cell. In analogy to epigenetics that describes "functionally relevant changes to the genome that do not involve a change in the nucleotide sequence", epitranscriptomics involves all functionally relevant changes to the transcriptome that do not involve a change in the ribonucleotide sequence. Thus, the epitranscriptome can be defined as the ensemble of such functionally relevant changes.

Protein methylation is a type of post-translational modification featuring the addition of methyl groups to proteins. It can occur on the nitrogen-containing side-chains of arginine and lysine, but also at the amino- and carboxy-termini of a number of different proteins. In biology, methyltransferases catalyze the methylation process, activated primarily by S-adenosylmethionine. Protein methylation has been most studied in histones, where the transfer of methyl groups from S-adenosyl methionine is catalyzed by histone methyltransferases. Histones that are methylated on certain residues can act epigenetically to repress or activate gene expression.

DNA methylation in cancer plays a variety of roles, helping to change the healthy cells by regulation of gene expression to a cancer cells or a diseased cells disease pattern. One of the most widely studied DNA methylation dysregulation is the promoter hypermethylation where the CPGs islands in the promoter regions are methylated contributing or causing genes to be silenced.

<span class="mw-page-title-main">Phasevarion</span>

In bacteria, phasevarions mediate a coordinated change in the expression of multiple genes or proteins. This occurs via phase variation of a single DNA methyltransferase. Phase variation of methyltransferase expression results in differential methylation throughout the bacterial genome, leading to variable expression of multiple genes through epigenetic mechanisms.

References

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  2. ↑ Hiraoka, Satoshi; Sumida, Tomomi; Hirai, Miho; Toyoda, Atsushi; Kawagucci, Shinsuke; Yokokawa, Taichi; Nunoura, Takuro (2021-05-08). "Diverse DNA modification in marine prokaryotic and viral communities". doi:10.1101/2021.05.08.442635. S2CID   234348602.{{cite journal}}: Cite journal requires |journal= (help)
  3. 1 2 Horton, John R.; Woodcock, Clayton B.; Opot, Sifa B.; Reich, Norbert O.; Zhang, Xing; Cheng, Xiaodong (2019-10-10). "The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site". Nature Communications. 10 (1): 4600. Bibcode:2019NatCo..10.4600H. doi:10.1038/s41467-019-12498-7. ISSN   2041-1723. PMC   6787082 . PMID   31601797.
  4. ↑ Schübeler, Dirk (January 2015). "Function and information content of DNA methylation". Nature. 517 (7534): 321–326. Bibcode:2015Natur.517..321S. doi:10.1038/nature14192. ISSN   1476-4687. PMID   25592537. S2CID   4403755.
  5. ↑ Vasu, K.; Nagaraja, V. (2013-03-01). "Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense". Microbiology and Molecular Biology Reviews. 77 (1): 53–72. doi:10.1128/MMBR.00044-12. ISSN   1092-2172. PMC   3591985 . PMID   23471617.
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  10. ↑ Staff, The PLOS Genetics (2016-05-12). "Correction: The Epigenomic Landscape of Prokaryotes". PLOS Genetics. 12 (5): e1006064. doi: 10.1371/journal.pgen.1006064 . ISSN   1553-7404. PMC   4865105 . PMID   27171000.
  11. 1 2 Gonzalez, Diego; Kozdon, Jennifer B.; McAdams, Harley H.; Shapiro, Lucy; Collier, Justine (April 2014). "The functions of DNA methylation by CcrM in Caulobacter crescentus: A global approach". Nucleic Acids Research. 42 (6): 3720–3735. doi:10.1093/nar/gkt1352. ISSN   1362-4962. PMC   3973325 . PMID   24398711.
  12. 1 2 Mouammine, Annabelle; Collier, Justine (October 2018). "The impact of DNA methylation in Alphaproteobacteria". Molecular Microbiology. 110 (1): 1–10. doi: 10.1111/mmi.14079 . ISSN   0950-382X. PMID   29995343.
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  17. ↑ Maier, Johannes A.H.; Albu, Razvan F.; Jurkowski, Tomasz P.; Jeltsch, Albert (December 2015). "Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM". Biochimie. 119: 60–67. doi:10.1016/j.biochi.2015.10.011. PMID   26475175.
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  19. ↑ Woodcock, Clayton B.; Yakubov, Aziz B.; Reich, Norbert O. (August 2017). "Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition". Biochemistry. 56 (30): 3913–3922. doi:10.1021/acs.biochem.7b00378. ISSN   0006-2960. PMID   28661661.
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