vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJGB-22-41vavilov-3383Research ArticleМОЛЕКУЛЯРНАЯ БИОЛОГИЯMOLECULAR BIOLOGYХарактеристика деметилирующей ДНК-гликозилазы ROS1 из Nicotiana tabacum L.Characterization of demethylating DNA glycosylase ROS1 from Nicotiana tabacum L.ПетроваД. В.PetrovaD. V.

Новосибирск

Novosibirsk

https://orcid.org/0000-0001-9291-1343ПермяковаН. В.PermyakovaN. V.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-5685-1248ГринИ. Р.GrinI. R.

Новосибирск

Novosibirsk

https://orcid.org/0000-0001-5013-0194ЖарковД. О.ZharkovD. O.

Новосибирск

Novosibirsk

dzharkov@niboch.nsc.ruИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наукРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of SciencesRussian FederationФедеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наукРоссияInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesRussian FederationИнститут химической биологии и фундаментальной медицины Сибирского отделения Российской академии наук; Новосибирский национальный исследовательский государственный университетРоссияInstitute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State UniversityRussian Federation202206072022264341348Copyright © Петрова Д.В., Пермякова Н.В., Грин И.Р., Жарков Д.О., 20222022Петрова Д.В., Пермякова Н.В., Грин И.Р., Жарков Д.О.Petrova D.V., Permyakova N.V., Grin I.R., Zharkov D.O.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/3383

Один из главных механизмов эпигенетической регуляции у высших эукариот основан на метилировании цитозина по положению C5 с образованием 5-метилцитозина (mC), который далее узнается регуляторными белками. У млекопитающих метилирование преимущественно протекает в динуклеотидах CG, тогда как у растений его мишенью служат последовательности CG, CHG и CHH (H – любое основание, кроме G). Корректное поддержание статуса метилирования ДНК требует баланса процессов метилирования, пассивного и активного деметилирования. В то время как у млекопитающих активное деметилирование происходит за счет направленного регулируемого повреждения mC в ДНК с последующим действием ферментов репарации, у растений функции деметилирования выполняют специализированные ДНК-гликозилазы, гидролизующие N-гликозидную связь mC-нуклеотидов. Геном модельного растения Arabidopsis thaliana кодирует четыре паралогичных белка, два из которых – DEMETER (DME) и REPRESSOR OF SILENCING 1 (ROS1) – обладают 5-метилцитозин-ДНК-гликозилазной активностью и необходимы для регуляции развития, ответа на инфекции и абиотический стресс и сайленсинга трансгенов и мобильных элементов. Гомологи DME и ROS1 присутствуют во всех группах растений, однако за пределами A. thaliana исследованы крайне слабо. В статье приведены результаты изучения свойств рекомбинантного фрагмента белка ROS1 из Nicotiana tabacum (NtROS1), содержащего основные структурные домены, необходимые для каталитической активности. Методами гомологичного моделирования была построена структурная модель NtROS1, в которой выявлена укладка, характерная для ДНК-гликозилаз структурного суперсемейства «спираль–шпилька–спираль». Рекомбинантный белок NtROS1 был способен удалять из ДНК основания mC, причем активность фермента слабо зависела от статуса метилирования CG-динуклеотидов в противоположной цепи. С меньшей эффективностью фермент удалял из ДНК 5-гидроксиметилцитозин (hmC), проявляя минимальную активность при наличии mC в противоположной цепи. При экспрессии гена NtROS1 в клетках человека в культуре происходило глобальное снижение уровня метилирования геномной ДНК. В целом можно сказать, что белок NtROS1 и другие гомологи DME и ROS1 представляют собой многообещающую основу для инженерии ферментов с целью анализа статуса эпигенетического метилирования и управления активностью генов.

One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of 5-methylcytosine (mC), which is further recognized by regulatory proteins. In mammals, methylation mainly occurs in CG dinucleotides, while in plants it targets CG, CHG, and CHH sequences (H is any base but G). Correct maintenance of the DNA methylation status is based on the balance of methylation, passive demethylation, and active demethylation. While in mammals active demethylation is based on targeted regulated damage to mC in DNA followed by the action of repair enzymes, demethylation in plants is performed by specialized DNA glycosylases that hydrolyze the N-glycosidic bond of mC nucleotides. The genome of the model plant Arabidopsis thaliana encodes four paralogous proteins, two of which, DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1), possess 5-methylcytosine-DNA glycosylase activity and are necessary for the regulation of development, response to infections and abiotic stress and silencing of transgenes and mobile elements. Homologues of DME and ROS1 are present in all plant groups; however, outside A. thaliana, they are poorly studied. Here we report the properties of a recombinant fragment of the ROS1 protein from Nicotiana tabacum (NtROS1), which contains all main structural domains required for catalytic activity. Using homologous modeling, we have constructed a structural model of NtROS1, which revealed folding characteristic of DNA glycosylases of the helix– hairpin–helix structural superfamily. The recombinant NtROS1 protein was able to remove mC bases from DNA, and the enzyme activity was barely affected by the methylation status of CG dinucleotides in the opposite strand. The enzyme removed 5-hydroxymethylcytosine (hmC) from DNA with a lower efficiency, showing minimal activity in the presence of mC in the opposite strand. Expression of the NtROS1 gene in cultured human cells resulted in a global decrease in the level of genomic DNA methylation. In general, it can be said that the NtROS1 protein and other homologues of DME and ROS1 represent a promising scaffold for engineering enzymes to analyze the status of epigenetic methylation and to control gene activity.

эпигенетическое деметилирование5-метилцитозин5-гидроксиметилцитозинДНК-гликозилазыREPRESSOR OF SILENCING 1Nicotiana tabacumepigenetic demethylation5-methylcytosine5-hydroxymethylcytosineDNA glycosylasesREPRESSOR OF SILENCING 1Nicotiana tabacumThe study was supported by the Russian Foundation for Basic Research and the Government of the Novosibirsk Region (project No. 20-44-540007). The modeling of the structure of NtROS1 was supported by the Russian Ministry of Science and Education (State budget project No. 121031300056-8). DNA sequencing was performed at the SB RAS Genomics Core Facility.ReferencesAgius F., Kapoor A., Zhu J.-K. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc. Natl. Acad. Sci. USA. 2006;103(31):11796-11801. DOI 10.1073/pnas.0603563103.Agius F., Kapoor A., Zhu J.-K. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc. Natl. Acad. Sci. USA. 2006;103(31):11796-11801. 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The authors declare that there are no conflicts of interest present.