vestnik-bio-msuВестник Московского университета. Серия 16. БиологияVestnik Moskovskogo universiteta. Seriya 16. Biologiya0137-0952Lomonosov Moscow State University, School of Biology10.55959/MSU0137-0952-16-80-3S-10vestnik-bio-msu-1543Research ArticleОригинальные исследованияPARP1-зависимые изменения нуклеосомной организации: возможное участие p53PARP1-dependent alterations in nucleosome organization: potential involvement of p53https://orcid.org/0000-0001-8137-4685КошкинаД. О.KoshkinaD. O.

Кошкина Дарья Олеговна – мл. науч. сотр. кафедры биоинженерии биологического факультета МГУ; ст. лаборант-исследователь отдела клеточной геномики группы динамики хроматина ИБГ РАН 

119234, г. Москва, Ленинские горы, д. 1, стр. 12

119334, г. Москва, ул. Вавилова, д. 34/5  

Тел.: 8-499-135-60-89 

 1–12 Leninskie gory, Moscow, 119234 

 34/5 Vavilov Str., Moscow, 119334 

koshkina.daria.2015@post.bio.msu.ruhttps://orcid.org/0000-0003-4129-4984МалюченкоН. В.MaluchenkoN. V.

Малюченко Наталия Валериевна – канд. биол. наук, доц. кафедры биоинженерии биологического факультета 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

Тел.: 8-495 939-57-38 

 1–12 Leninskie gory, Moscow, 119234 

mal_nat@mail.ruНовичковаА. М.NovichkovaA. M.

Новичкова Антонина Михайловна – студентка кафедры молекулярного биологии биологического факультета 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

Тел.: 8-495-939-57-38 

 1–12 Leninskie gory, Moscow, 119234 

tonya.novichkova@bk.ruhttps://orcid.org/0000-0002-1596-9506ФеофановА. В.FeofanovA. V.

Феофанов Алексей Валерьевич – докт. биол. наук, проф. кафедры биоинженерии биологического факультета МГУ; вед. науч. сотр. отдела клеточной геномики группы динамики хроматина ИБГ РАН 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

Тел.: 8-499-135-60-89 

 1–12 Leninskie gory, Moscow, 119234

 34/5 Vavilov Str., Moscow, 119334  

avfeofanov@yandex.ruhttps://orcid.org/0000-0002-7389-7993СтудитскийВ. М.StuditskyV. M.

Студитский Василий Михайлович – докт. биол. наук, вед. науч. сотр. кафедры биоинженерии биологического факультета 

119234, г. Москва, Ленинские горы, д. 1, стр. 12 

333 Cottman Ave., Philadelphia, 19111, Pennsylvania 

Тел.: 8-495-939-57-38 

 1–12 Leninskie gory, Moscow, 119234 

 333 Cottman Ave., Philadelphia, 19111, Pennsylvania 

vasily.studitsky@fccc.eduБиологический факультет, Московский государственный университет имени М.В. Ломоносова; Институт биологии гена, Российская академия наукРоссияSchool of Biology, Lomonosov Moscow State University; Institute of Gene Biology, Russian Academy of SciencesRussian FederationБиологический факультет, Московский государственный университет имени М.В. ЛомоносоваРоссияSchool of Biology, Lomonosov Moscow State UniversityRussian FederationБиологический факультет, Московский государственный университет имени М.В. Ломоносова; Fox Chase Cancer CenterРоссияSchool of Biology, Lomonosov Moscow State University; Fox Chase Cancer CenterRussian Federation202510112025803S6772Copyright © Кошкина Д.О., Малюченко Н.В., Новичкова А.М., Феофанов А.В., Студитский В.М., 20252025Кошкина Д.О., Малюченко Н.В., Новичкова А.М., Феофанов А.В., Студитский В.М.Koshkina D.O., Maluchenko N.V., Novichkova A.M., Feofanov A.V., Studitsky V.M.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik-bio-msu.elpub.ru/jour/article/view/1543

Хроматин эукариот представляет собой высокоорганизованную и динамичную структуру, состоящую из ДНК и ассоциированных белков. Эти белки обеспечивают точную регуляцию ключевых процессов, включая экспрессию генов, репликацию и репарацию ДНК. Важнейшими регуляторами архитектуры хроматина из негистоновых белков, являются p53 и PARP1, которые участвуют в ответе клетки на повреждения ДНК. В настоящей работе проведено исследование кооперативного и конкурентного связывания ДНК-связывающего домена (DBD, DNA-Binding Domain) белка p53, и фермента PARP1 с мононуклеосомами, реконструированными на основе последовательности Widom 603 с встроенным сайтом связывания p53. Для детекции взаимодействий использован метод электрофоретического сдвига подвижности (EMSA, Electrophoretic Mobility Shift Assay) с флуоресцентно меченными нуклеосомами. Комплексы формировали двумя способами: предварительно инкубировали нуклеосомы с p53 DBD и затем добавляли PARP1, либо сначала получали комплекс нуклеосома–PARP1 и затем вносили p53 DBD. Результаты показали, что порядок добавления белков определяет характер их взаимодействия с нуклеосомой: при низких концентрациях p53 наблюдается вытеснение этого белка PARP1, тогда как при повышении концентрации p53 формируются стабильные комплексы нуклеосома–p53, не нарушенные белком PARP1. Стабильных тройных комплексов нуклеосома–p53–PARP1 не обнаружено.

Chromatin of eukaryotic organisms is a complexly organized and dynamic complex. Chromatin proteins provide proper regulation of gene expression, DNA replication, and DNA repair. Among the most important regulators of chromatin architecture among non-histone proteins are p53 and PARP1, which are involved in the cellular response to DNA damage. In the present study, we investigated the cooperative and competitive binding of the DNA-binding domain (DBD) of p53 and the enzyme PARP1 to mononucleosomes reconstituted on the Widom 603 sequence with an embedded p53 binding site. To detect interactions, the electrophoretic mobility shift assay (EMSA) method with fluorescently labeled DNA. Complexes were formed in two ways: nucleosomes were pre-incubated with p53 DBD and then PARP1 was added, or the nucleosome–PARP1 complex was formed first and then p53 DBD was introduced. The results showed that the order of protein addition determines the nature of their interaction with the nucleosome: at low p53 concentrations, displacement of this protein by PARP1 is observed, while at higher p53 concentrations, stable nucleosome–p53 complexes are formed, undisturbed by PARP1. No stable ternary nucleosome–p53–PARP1 complexes were detected.

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The authors declare that there are no conflicts of interest present.