Gla I

Description

Recognition site and hydrolysis position:

Source: An E.coli strain that carries the cloned Gla I gene from Glacial ice bacterium Gl29
Substrate specificity:
Unit definition: One unit is defined as the amount of enzyme required to hydrolyse completely a unique 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` site in 1 μg of pHspAI2 plasmid DNA, which is linearized with GsaI, in 1 hour at 30°C in a total reaction volume of 50 μl. As a result of this site hydrolysis two main DNA fragments (3,419 and 699 bp) are produced (see lanes 3-5 in the figure). GlaI digestion of recognition sequences with three and two 5-methylcytosines results in additional bands appearance (lane 6 in the figure).

GlaI activity assay on DNA pHspAI2/GsaI Lanes: 2 Control pHspAI2/GsaI DNA 3 – 0.5 μl GlaI (diluted 1/100), 4 – 1 μl GlaI (diluted 1/100), 5 – 2 μl GlaI (diluted 1/100), 6 – 1 μl of undiluted GlaI, 1 and 7- 1 Kb SE DNA Ladder.Products were separated in 1% agarose gel in TAE Buffer.

In the presence of 20% DMSO, the enzyme’s activity significantly increases without losing specificity

Assayed on: DNA pHspAI2/GsaI is a linearized plasmid pHspAI2, which carries a gene of DNA-methyltransferase M.HspAI (recognition sequence 5`-GCGC-3`) and includes a unique GlaI recognition site 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` [2].
Optimal SE-buffer: SE-buffer Y
Enzyme activity (%):

Storage conditions: 10 mM Tris-HCl (pH 7.6); 250 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,1% Triton X-100, 0.05 mg/ml BSA, 50% glycerol; Store at -20°C.
Ligation: –
Non-specific hydrolisis: No detectable degradation of 1μg of Lambda DNA was observed after incubation with 100 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.Reagents Supplied with Enzyme

10 X SE-buffer Y, pHspAI2/GsaI DNA
Methylation sensitivity: The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNAand DNA with N4-methylcytosines [1].
Notes:

References:

1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium I – producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2006).
2. Valery A. Chernukhin, Tatyana N. Najakshina, Murat A. Abdurashitov, Julia E. Tomilova, Nina V. Mezentzeva, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Danila A. Gonchar, Sergei Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5’-G(m5C)^GC-3’ // Biotechnologia (russ.). 2006. N 4. P. 31-35
3. Degtyarev S.Kh., Belavin P.A., Repin V.E., Malygin E.G. Preparation method of restriction endonuclease Fok I from Flavobacterium okeanokoites // Soviet Union patent SU1436160 (1988). (In Russian)
4. Degtyarev S.Kh., Rechkunova N.I., Grinev A.A., Dedkov V.S. Discovery and substrate specificity determination of restriction endonucleases Bme18I and Kzo9I.// Izvestia SB SA USSR, Biological sciences series, No.3, 25-26 (1989). (In Russian)
5. Zemlyanskaya, E.V., Degtyarev, S.K. Substrate specificity and properties of methyl-directed site-specific DNA endonucleases.// Molecular Biology, v.47, No.6, p.900-913 (2013)
   

Application:

• L. Zhen, X. Tang, Z. Xu, Y. Huang, X. Qian, H. Lin, C. Li, R. Cui, H. Fang, H. Yang, J. Qiu, Z. Fang, X. Peng, Y. Jin, J. Nie, S. Guo, Y. Wang, M. Zhong, H. Gu, H. Xu, Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility. Adv. Sci. 2024, 2401137. https://doi.org/10.1002/advs.202401137
• Qiaomin Wu, Yang Yu, Mengqi Chen, Jinyan Long, Xiaolan Yang A label-free fluorescence sensing strategy based on GlaI-assisted EXPAR for rapid and accurate quantification of human methyltranferase activity // Talanta, 269, 125456 (2024)
•  Xu, G., Yang, H., Qiu, J. et al. Sequence terminus dependent PCR for site-specific mutation and modification detection.// Nature Communications 14, 1169 (2023)
•  Zhou S, Sun H, Huo D, Wang X, Qi N, Peng L, Yang M, Lu P, Hou C.A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation.// Analytica Chimica Acta, Volume 1212, 15 June 2022
• Hao Yang, Jiani Qiu, LinQing Zhen, Yizhou Huang, Wei Ren, Hongchen Gu, Hong Xu, Gaolian Xu.Sensitive GlaI digestion and terminal transferase PCR for DNA methylation detection// Talanta 2022 Sep 1;247:123616 (2022)
•  Dong N, Wang W, Qin Y, Wang Y, Shan H.Sensitive lateral flow assay for bisulfite-free DNA methylation detection based on the restriction endonuclease GlaI and rolling circle amplification// Analytica Chimica Acta, Volume 1227, 22 September 2022
•  Wang LJ, Han X, Qiu JG, Jiang B, Zhang CY.Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level// Chem Sci. 2020, 11, 9675-9684 (2020)
• Petrova, D. V., Naumenko, M. B., Khantakova, D. V., Grin, I. R. & Zharkov, D. O. Relative Efficiency of Recognition of 5-Methylcytosine and 5-Hydroxymethylcytosine by Methyl-Dependent DNA Endonuclease GlaI// Russian Journal of Bioorganic Chemistry T. 45, № 6, стр. 625-629. (2019)
• Susan M Mitchell, Keith N Rand, Zheng-Zhou Xu, Thu Ho, Glenn S Brown, Jason P Ross, Peter L Molloy Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences // Methods Mol Biol (2018)
•  Yueying Sun, Yuanyuan Sun, Weimin Tian, Chenghui Liu, * Kejian Gao and Zhengping L A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction // Chemical Science, 9, pp.1344-1351 (2018)
•  Viktor Tomilov, Murat Abdurashitov, Danila Gonchar, Anastasiya Snezhkina, George Krasnov, Anna Kudryavtseva and Sergey Degtyarev Comparative analysis of RCGY sites methylation in the genomes of Raji and U-937 malignant and normal lung fibroblast cell lines // Cancer Epigenetics and Biomarkers, Osaka, Japan, October 2017
•  A.G. Akishev et al. GLAD-PCR assay of selected R(5mC)GY sites in URB1 and CEPBD genes in human genome// Res J Pharm Biol Chem Sci, 8(1): pp.465-475, (2017)
•  Evdokimov et al. GLAD-PCR Assay of DNA Methylation Markers Associated with Colorectal Cancer// Biol Med (Aligarh) , 8:7(2016)
•  A.A. Evdokimov, N.A. Netesova, N.A. Smetannikova, M.A. Abdurashitov, A.G. Akishev, E.S. Davidovich, Yu.D. Ermolaev, A.B. Karpov, A.E. Sazonov, R.M. Tahauov, S.Kh. Degtyarev Application of GLAD-PCR assay for determination of the methylation sites in the regulatory regions of tumor-supressors gene ELMO1 and ESR1 in colorectal cancer// Translated from Problems in oncology, #1, p.116-120 (2016).
•  EV Dubinin, AG Akishev, MA Abdurashitov, SB Oleynikova, VL Sitko, and S Kh Degtyarev Real time GlaI-PCR assay of regulation regions of human genes HDAC4, RARB and URB1 // Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol 7(2), p.p. 667-676 (2016).
•  Murat A . Abdurashitov , Valery A . Chernukhin , Danila A . Gonchar , Vladimir S . Dedkov, Natalia A . Mikhnenkova, and Sergey Kh . Degtyarev Dimethyl Sulfoxide Changes the Recognition Site Preference of Methyl- directed Site-specific DNA Endonuclease GlaI// Research Journal of Pharmaceutical, Biological and Chemical Sciences, № 7(1),с. 1733-1739 (2016)
•  Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Kuznetsov V.V., Degtyarev S.Kh. Mapping of R(5mC)GY Sites in the Genome of Human Malignant Cell Line Raji // Biol Med (Aligarh) Volume 7, Issue 4, BM-135-15 (2015)
• Kuznetsov V.V., Abdurashitov M.A., Akishev A.G., Degtyarev S.H. Method for detection nucleotide sequence R(5mC)GY in given position of continuous DNA // Russian Federation patent RU 2525710 C1 (2014).
• Fagan RL, Wu M, Chedin F, Brenner C An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes //PLoS ONE 8(11): e78752. doi:10.1371/journal.pone.0078752 (2013)
• Keith N. Rand, Graeme P. Young, Thu Ho and Peter L. Molloy, Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylationdependent restriction enzymes. // Nucleic Acids Research, pp. 1-10 (2012).
• F. Syeda, R.L. Fagan, M. Wean, G.V. Awakumov, J.R. Walker, S. Xue, S. Dhe-Paganon, & C. Brenner, The RFTS Domain is a DNA-competitive Inhibitor of Dnmt1//, JBC, v. 286, pp. 15344-15351 (2011).
• Kravets AP, Mousseau TA, Litvinchuk AV, Ostermiller Sh, Vengen GS, Grodzinski DM. Changes in wheat DNA methylation pattern after chronic seed gamma-irradiation.// Tsitol Genet. 2010 Sep-Oct;44(5):18-22. Russian.
• Wood, R. J., McKelvie, J. C., Maynard-Smith, M. D., and Roach, P. L. A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.// (2010) Nucleic Acids Res 38, e107
• Abdurashitov M.A., Chernukhin V.A, Gonchar D.A., Degtyarev S.Kh. GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation // BMC Genomics 2009, 10:322.