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Article Evaluation

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1. For the most part everything in the article is relevant to the topic, and sub topics regarding society, culture, and public policy which aren't directly related to CRISPR as a biological complex are blended in nicely by discussing the current and potential ramifications/controversies.

2. The article appears to be neutral and accomplishes this by a significant discussion of the biological and mechanistic mechanisms and any discussion of controversies is not left without discussion of it's scientific merit and positive recognition by the scientific community. Overall this article is heavy in science and cites a lot of peer reviewed articles.

3. The article has an extensive set of points which it discusses but I noticed that it focused heavily on the applications that have been achieved or are being attempted currently and there is no section on limitations asides from policies and regulations. It would be nice for a reader to know given current technologies what can CRISPR not do in todays world that it could do in a "perfect world".

4. Yes from checking about 5-6 superscript paper links, they work and the publications cited to make statements are valid.

5. Nothing is really out of date as most of the literature citation appears to be using material from 2011-2017 and significant discoveries in 2017 have been included. As a novice biochemist I think that because this page has a short section on structure and images of CRISPR there should be inclusion of studies into the structure/function of the CRISPR complex. This would be an interesting read for structural biologists/biochemists who are generally inclined to make mutants.

6. Surprisingly the talk page for this article is empty?? CRISPR is the hottest topic right now in all bio related fields.

7. I am not sure how this article is rated, I could not find any rating description.

In the brain

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The activity of DNA methylation has been shown to change during periods of memory consolidation in the rat hippocampus[1], a brain region important for memory formation [2]. Evidence of a role for DNA methylation in regulating Synaptic plasticity has been shown through inhibition of mice cytosine methyltransferases which blocked the long term potentiation at Schaffer collateral synapses[3], synaptic plasticity has been touted to be a foundation for memory formation as found in the Hebbian theory.

Dam methylase

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It is an orphan methyltransferase that is not part of a restriction-modification system and regulates gene expression [4].

Role in regulation of protein expression

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Given it's role of protein regulation in E. coli, the Dam methylase gene is nonessential as a knockout of the gene still leaves the bacteria viable[5]. The retainment of viability despite a dam gene knockout is also seen in Salmonella sp.[6] and Aggregatibacter actinomycetemcomitans [7]. However in organisms like Vibrio cholerae and Yersinia pseudotuberculosis, the dam gene is essential for viability[8]. A knockout of the dam gene in Aggregatibacter actinomycetemcomitans resulted in dysregulated levels of the protein, leukotoxin, and also reduced the microbe's ability to invade oral epithelial cells[7]. Additionally a study on Dam methylase deficient Streptococcus mutans, a dental pathogen, revealed the dysregulation of a 103 genes some of which include cariogenic potential.

Structural Features of Dam Methylase

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The similarity in the catalytic domains of C5-cytosine methyltransferases and N6 and N4-adenine methyltransferases provided great interest in understanding the basis for functional similarities and dissimilarities. The methyltransferases or methylases are classified into 3 groups (Groups α, β, and γ) based on the sequential order of certain 9 motifs and the Target Recognition Domain (TRD)[9]. Motif I consists of a Gly-X-Gly tripeptide and is referred to as the G-loop and is implicated in the binding of the S-Adenosyl methionine cofactor[10]. Motif II is highly conserved among N4 and N6-adenine methylases and contains a negatively charged amino acid followed by a hydrophobic side chain in the last positions of the β2 strand to bind the AdoMet[9]. Motif III is also implicated in the binding of Adomet. Motif IV is especially important and well known in methylase characterizations. It consists of a diprolyl component and is highly conserved among N6-adenine methyltransferases as the DPPY motif, however, this motif can vary for N4-adenine and C5-cytosine methyltransferases. The DPPY motif has been found to be essential for AdoMet binding[11]. Motifs IV-VIII play a role in the catalytic activity, while motifs 1-III and X play a role in binding of the cofactor. For N6-adenine methylases the sequential order for these motifs is as such: N-terminal - X - I - II - III - TRD - IV - V - VI - VII - VIII - C-terminal and E. coli Dam methylase follows this structural sequence [9]. A recent crystallography experiment showed that E. coli Dam methylase was able to bind non-GATC DNA with the same sequence of motifs discussed, the authors posit that the obtained structure could serve as grounds for repression of transcription that is not methylation based[12].

An X-ray crystal structure shows Escherichia coli Dam methylase bound to double stranded DNA and the inhibitor sinefungin.
The X-ray crystal structure of E. coli Dam methylase shows the enzyme bound to double stranded DNA and the inhibitor sinefungin. The adenine to be modified is shown as a blue stick flipped out of the double helix and towards the enzyme's interior.

Orphan and Phage methyltransferases

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Dam methylase is an orphan methyltransferase that is not part of a restriction-modification system but operates independently to regulate gene expression, mismatch repair, and bacterial replication amongst many other functions. This is not the only example of an orphan methyltransferase as there exists the Cell cycle regulated methyltransferase (CcrM) which methylates 5'-GANTC-'3 hemi-methylated DNA to control the life cycle of Caulobacter crescentus and other related species[13].

Distinct from their bacterial counterparts, phage orphan methyltransferases also do exist and most notably in the T2, T4, and other T-even bacteriophages that infect E. coli [4]. In a study it was identified that despite sharing any sequence homology, the E. coli and T4 Dam methylase amino acids sequences share sequence identity of up to 64 % in four regions of 11 to 33 residues long which suggests a common evolutionary origin for the bacterial and phage methylase genes[14]. The T2 and T4 methylases differ from E. coli Dam methylase in not only their ability to methylate 5-hydroxymethylcytosine but to also methylate non-canonical DNA sites[15]. Despite extensive in vitro characterization of these select few phage orphan methyltransferases their biological purpose is still not clear[4].

  1. ^ Lubin, F. D.; Roth, T. L.; Sweatt, J. D. (15 October 2008). "Epigenetic Regulation of bdnf Gene Transcription in the Consolidation of Fear Memory". Journal of Neuroscience. 28 (42): 10576–10586. doi:10.1523/JNEUROSCI.1786-08.2008.
  2. ^ Morris, R. G. M.; Garrud, P.; Rawlins, J. N. P.; O'Keefe, J. (24 June 1982). "Place navigation impaired in rats with hippocampal lesions". Nature. 297 (5868): 681–683. doi:10.1038/297681a0.
  3. ^ Levenson, Jonathan M.; Roth, Tania L.; Lubin, Farah D.; Miller, Courtney A.; Huang, I-Chia; Desai, Priyanka; Malone, Lauren M.; Sweatt, J. David (9 June 2006). "Evidence That DNA (Cytosine-5) Methyltransferase Regulates Synaptic Plasticity in the Hippocampus". Journal of Biological Chemistry. 281 (23): 15763–15773. doi:10.1074/jbc.M511767200.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  4. ^ a b c Murphy, James; Mahony, Jennifer; Ainsworth, Stuart; Nauta, Arjen; Sinderen, Douwe van (2013-12-15). "Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin, Function, and Occurrence". Applied and Environmental Microbiology. 79 (24): 7547–7555. doi:10.1128/aem.02229-13. ISSN 0099-2240. PMID 24123737.
  5. ^ Bale, Allen; d'Alarcao, Marc; Marinus, M.G. "Characterization of DNA adenine methylation mutants of Escherichia coli K12". Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 59 (2): 157–165. doi:10.1016/0027-5107(79)90153-2.
  6. ^ Nicholson, Brad; Low, David (2000-02-01). "DNA methylation-dependent regulation of Pef expression in Salmonella typhimurium". Molecular Microbiology. 35 (4): 728–742. doi:10.1046/j.1365-2958.2000.01743.x. ISSN 1365-2958.
  7. ^ a b Wu, H.; Lippmann, J. E.; Oza, J. P.; Zeng, M.; Fives-Taylor, P.; Reich, N. O. (2006-08-01). "Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans". Oral Microbiology and Immunology. 21 (4): 238–244. doi:10.1111/j.1399-302x.2006.00284.x. ISSN 1399-302X.
  8. ^ Julio, Steven M.; Heithoff, Douglas M.; Provenzano, Daniele; Klose, Karl E.; Sinsheimer, Robert L.; Low, David A.; Mahan, Michael J. (2001-12-01). "DNA Adenine Methylase Is Essential for Viability and Plays a Role in the Pathogenesis of Yersinia pseudotuberculosis andVibrio cholerae". Infection and Immunity. 69 (12): 7610–7615. doi:10.1128/iai.69.12.7610-7615.2001. ISSN 0019-9567. PMID 11705940.
  9. ^ a b c Malone, Thomas; Blumenthal, Robert M.; Cheng, Xiaodong (November 1995). "Structure-guided Analysis Reveals Nine Sequence Motifs Conserved among DNA Amino-methyl-transferases, and Suggests a Catalytic Mechanism for these Enzymes". Journal of Molecular Biology. 253 (4): 618–632. doi:10.1006/jmbi.1995.0577.
  10. ^ Schluckebier, Gerd; O'Gara, Margaret; Saenger, Wolfram; Cheng, Xiaodong. "Universal Catalytic Domain Structure of AdoMet-dependent Methyltransferases". Journal of Molecular Biology. 247 (1): 16–20. doi:10.1006/jmbi.1994.0117.
  11. ^ Kossykh, Valeri G.; Schlagman, Samuel L.; Hattman, Stanley (1993-07-25). "Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N 6 -adenine]-methyltransferase is important for S-adenosyl-L-methionine binding". Nucleic Acids Research. 21 (15): 3563–3566. doi:10.1093/nar/21.15.3563. ISSN 0305-1048.
  12. ^ Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; Cheng, Xiaodong (2015-04-30). "Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression". Nucleic Acids Research. 43 (8): 4296–4308. doi:10.1093/nar/gkv251. ISSN 0305-1048.
  13. ^ Zweiger, Gary; Marczynski, Gregory; Shapiro, Lucille. "A Caulobacter DNA Methyltransferase that Functions only in the Predivisional Cell". Journal of Molecular Biology. 235 (2): 472–485. doi:10.1006/jmbi.1994.1007.
  14. ^ Hattman, Stanley (August 6 1985). "Common Evolutionary Origin of the Phage T4 dam and Host Escherichia coli dam DNA-Adenine Methyltransferase Genes" (PDF). Journal of Bacteriology. 164: 932–937 – via Journal of Bacteriology. {{cite journal}}: Check date values in: |date= (help); line feed character in |title= at position 56 (help)
  15. ^ Schlagaman, Samuel L.; Hattman, Stanley (1989-11-25). "The bacteriophage T2 and T4 DNA-[N6-adenine] methyltransferase (Dam) sequence specificities are not identical". Nucleic Acids Research. 17 (22): 9101–9112. doi:10.1093/nar/17.22.9101. ISSN 0305-1048.
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