Влияние повреждений остатков гуанина в ДНК на функционирование ДНК-метилтрансфераз SssI (Spiroplasma) и Dnmt3a (мыши) тема диссертации и автореферата по ВАК РФ 02.00.10, кандидат химических наук Мальцева, Диана Васильевна

• Мальцева, Диана Васильевна
• кандидат химических наук
• 2009

Оглавление диссертации кандидат химических наук Мальцева, Диана Васильевна

Список сокращений.

Введение.

Глава 1. Повреждения остатков гуанина в ДНК (Обзор литературы).

1.1. Повреждение ДНК активными формами кислорода.

1.1.1. Структура ДНК, содержащей 8-oxoG.

1.1.2. Репарация 8-охоО-ДНК.

1.1.3. Влияние 8-oxoG на процессы, протекающие в клетке.

1.2. Повреждение ДНК метилирующими агентами.

1.2.1. Структура ДНК, содержащей 06meG.

1.2.2. Ответ клетки на присутствие 06meG в ДНК.

1.2.3. Цитотоксичность 06meG.

1.3. Действие на ДНК бензо[а]пирена — канцерогенного полициклического ароматического углеводорода.

1.3.1. Структура ДНК, содержащей (+)-транс- и (+)-^ис-В[а]Р-Д,'2-сЮ-аддукты.

1.3.2. Репарация В[а]Р-ДНК.

1.3.3. Взаимодействие В[а]Р-ДНК с ДНК-связывающими ферментами.

Глава 2. Влияние повреждений остатков гуанина в ДНК на функционирование ДНК-метилтрансфераз SssI (Spiroplasma) и Dnmt3a (мыши) (Обсуждение результатов).

2.1. Взаимодействие Dnmt3a-CD и M.SssI с ДНК, содержащей 8-oxoG.

2.1.1. Выделение Dnmt3a-CD и M.SssI.

2.1.2. Дизайн и характеристика аналогов субстратов.

2.1.3. Начальные скорости метилирования Dnmt3a-CD ДНК-дуплексов, содержащих 8-oxoG.

2.1.4. Связывание Dnmt3a-CD с ДНК-дуплексами, содержащими 8-oxoG.

2.1.5. Интегральная кинетика метилирования Dnmt3a-CD ДНК-дуплексов, содержащих 8-oxoG.

2.1.6. Метилирование M.SssI ДНК-дуплексов, содержащих 8-oxoG.

2.1.7. Молекулярные основы влияния 8-oxoG на взаимодействие

Dnmt3a-CD и M.SssI с ДНК.

2.2. Взаимодействие Dnmt3a-CD и M.SssI с ДНК, содержащей 06meG.

2.2.1. Используемые ДНК-дуплексы.

2.2.2. Начальные скорости метилирования Dnmt3a-CD ДНК-дуплексов, содержащих 06meG.

2.2.3. Связывание Dnmt3a-CD с ДНК-дуплексами, содержащими 06meG.

2.2.4. Интегральная кинетика метилирования Dnmt3a-CD ДНК-дуплексов, содержащих 06meG.

2.2.5. Метилирование M.SssI ДНК-дуплексов, содержащих 06meG.

2.2.6. Молекулярные основы влияния 06meG на взаимодеиствие

Dnmt3a-CD и M.SssI с ДНК.

2.3. Взаимодействие M.SssI с ДНК, содержащей (+)-цис- B[«]P-iV2-dG.

2.3.1. Связывание M.SssI с ДНК-дуплексами, содержащими (+)-i{uc-B[a]P-N -dG.

2.3.2. Метилирование M.SssI ДНК-дуплексов, содержащих (+)-цис- B[a]P-./V -dG.

2.3.3. Сравнение влияния (+)-цис- и (+)-трсшс-В[а\Р-Аг -dG-аддуктов на функционирование M.SssI.

Введение диссертации (часть автореферата) на тему «Влияние повреждений остатков гуанина в ДНК на функционирование ДНК-метилтрансфераз SssI (Spiroplasma) и Dnmt3a (мыши)»

Клеточная ДНК постоянно повергается воздействию веществ, содержащихся в окружающей среде или образующихся в процессе естественного метаболизма, способных вызывать её повреждение. Это способствует возникновению мутаций и нарушению функционирования различных систем клетки.

Мишенями для повреждающих агентов во многих случаях служат остатки гуанина. Основным источником эндогенных повреждений гуанина являются активные формы кислорода, приводящие преимущественно к образованию 7,8-дигидро-8-оксогуапипа (8-oxoG) — биомаркера окислительного стресса. Взаимодействие ДНК с некоторыми продуктами катаболизма, а также метилирующими агентами, содержащимися в окружающей среде, приводит к появлению в ДНК очень токсичного для клетки О -метилгуанина (06meG). Среди загрязнителей окружающей среды одним из наиболее распространённых является бензо[а]пирен (В[а]Р). В процессе метаболизма в эукариотических клетках В[д]Р активируется до реакционноспособных диолэпоксидов, которые ковалентно присоединяются к ДНК по экзоциклической аминогруппе гуанина с преимущественным образованием (+)-цис- и (+)-/ирш;с-стереоизомерных B[a]P-A^-dG-аддуктов.

Действие окислительных и метилирующих агентов и В[а]Р на остатки гуанина в ДНК клеток млекопитающих может нарушить процесс ферментативного метилирования CG-последовательностей ДНК по атому углерода С5 остатков цитозина. Метилирование ДНК играет важную роль в регуляции многих клеточных процессов, в том числе транскрипции генов. Изменение активности генов, часто ассоциированное с аберрантным метилированием ДНК, является фактором, запускающим развитие многих заболеваний, в числе которых рак, диабет, заболевания сердечно-сосудистой системы. В клетках животных и человека метилирование CG-последовательностей в ДНК осуществляется С5-ДНК-метилтрансферазами (МТазами) Dnmtl, Dnmt3a и Dnmt3b. Эти ферменты обладают сложной структурой, а молекулярные механизмы их действия ещё мало изучены.

Показано, что все три МТазы важны как для установления, так и для поддержания нормального уровня метилирования ДНК. Представляется важным изучение влияния повреждений ДНК, приводящих к образованию 8-oxoG, 06meG и на функционирование МТаз.

Объектом исследования в настоящей работе стал каталитический домен Dnmt3a мыши (Dnmt3a-CD). В качестве второго объекта была выбрана прокариотическая МТаза SssI (M.SssI), которая, как и эукариотические ферменты, узнает в ДНК короткую CG-последовательность и традиционно используется в качестве модельного фермента при изучении эукариотических МТаз.

Целью работы явилось определение влияния повреждений остатков гуанина в ДНК на функционирование каталитического домена Dnmt3a мыши и M.SssI из Spiroplasma.

В работе решались следующие задачи: 1) исследование влияния 8-oxoG, 06meG и (+)-цис-В [a] P-A^-dG на различные стадии реакции метилирования ДНК каталитическим доменом Dnmt3a и M.SssI и 2) определение возможных молекулярных механизмов действия повреждений остатков гуанина в ДНК на функционирование Dnmt3a-CD и M.SssI.

Заключение диссертации по теме «Биоорганическая химия», Мальцева, Диана Васильевна

Выводы

1. Впервые показано, что связывание ДНК-метилтрансферазы Dnmt3a-CD с ДНК имеет кооперативный характер. Замена остатка гуанина в метилируемой цепи в участке узнавания или рядом с ним на 8-oxoG или 06meG вызывает снижение сродства Dnmt3a-CD к ДНК и увеличение кооперативности связывания.

2. Скорость метилирования ДНК МТазой Dnmt3a-CD зависит от локализации и характера повреждения остатка гуанина: в случае 8-oxoG наблюдается значительное замедление метилирования, а в случае 06meG — незначительное замедление или даже ускорение.

3. Dnmt3a-CD образует с ДНК как продуктивный, так и непродуктивный комплексы. Введение 8-oxoG и 06meG в ДНК практически не влияет на константу скорости реакции метилирования в условиях «одного оборота», однако приводит к изменению соотношения продуктивного и непродуктивного комплексов, что и объясняет наблюдаемое влияние повреждений на метилирование ДНК МТазой Dnmt3a-CD.

4. Замена остатка гуанина в участке узнавания на 8-oxoG или 06meG приводит к замедлению или блокированию метилирования ДНК МТазой SssI.

5. Влияние повреждений остатков гуанина в ДНК бензо[а]пиреном на функционирование M.Sssl определяется стереохимией В[я]Р-/У2-сЮ-аддуктов. В случае (+)-г/нс-изомера, когда остаток В[а]Р иптреркалирует в двойную спираль, способность МТазы SssI связывать и метилировать ДНК практически не изменяется, тогда как в случае (+)-транс-изомера, когда остаток В[а]Р расположен в малой бороздке, наблюдается блокирование метилирования.

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