Structural studies of proteins involved in DNA repair and recombination

参与 DNA 修复和重组的蛋白质的结构研究

• 批准号: 10919414
• 负责人: MARTIN F. GELLERT
• 金额: $ 138.1万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10919414
• 关键词: Adaptive Immune System; Binding; Biochemical; C-terminal; Chordata; Chromatin; Cryoelectron Microscopy; DNA; DNA Repair; DNA-dependent protein kinase; Ephrin-A5; Eukaryota; Exonuclease; Genes; Genetic Recombination; Histones; Human; Immunoglobulins; Lysine; Methylation; Multiprotein Complexes; Nonhomologous DNA End Joining; Process; Proteins; Rag1 Mouse; Reporting; Role; Shark; Site; Structure; T-Cell Receptor Genes; V(D)J Recombination; Work; cofactor; histone modification; homologous recombination; protein complex; protein structure

Continuing our work on the initiation of V(D)J recombination by the RAG1/2 protein complex, we have obtained a more extended protein structure containing the full C-terminal of RAG2 in addition to the catalytically active parts of RAG1 and RAG2 previously reported. This C-terminal extension contains a PHD domain that binds to a particular histone modification: histone 3 modified by trimethylation on Lysine 4 (known as H3K4Me) that characterizes active chromatin. The PHD domain thus helps to bring RAG1/2 to its desired sites of action. In addition, the PHD domain, when free of histone, inhibits RAG1/2 cleavage of DNA. Thus, the PHD domain pays a dual role in regulating V(D)J recombination. Our new structure makes it clear how this inhibition occurs. Continuing our work on the DNA-dependent protein kinase (DNA-PK), we are now looking at its role in initiating homologous recombination. This is known to involve its interaction with the three-protein complex of Mre11, Rad50, and Nbs 1 (MRN), which has both endo- and exonuclease activity near a DNA-PK blocked DNA end, thus creating the necessary 3 overhang on DNA. This challenging multi-protein complex (seven proteins altogether, including cofactors) is being studied by cryo-electron microscopy.

继续我们对RAG1/2蛋白复合物进行V（d）J重组的工作，除了先前报道的RAG1和RAG2的催化活性部分外，我们还获得了更扩展的蛋白质结构，该结构含有RAG2的完整C末端。该C末端扩展包含一个与特定组蛋白修饰结合的PHD结构域：组蛋白3通过赖氨酸4（称为H3K4ME）在表征活性染色质的赖氨酸4（称为H3K4ME）上修饰。因此，PHD域有助于将RAG1/2带到所需的作用位置。另外，当不含组蛋白的PHD结构域抑制DNA的RAG1/2裂解。因此，PHD结构域在调节V（d）J重组中起着双重作用。我们的新结构清楚地表明了这种抑制是如何发生的。 继续我们在DNA依赖性蛋白激酶（DNA-PK）上的工作，我们现在正在研究其在启动同源重组中的作用。众所周知，这涉及其与MRE11，Rad50和NBS 1（MRN）的三蛋白复合物的相互作用，该复合物在DNA-PK阻断的DNA端附近具有内核酸酶和外切核酸酶的活性，从而在DNA上产生了必要的3个悬垂性。这种具有挑战性的多蛋白质复合物（包括辅助剂在内的七个蛋白质）正在通过冷冻电子显微镜研究。

项目成果

Structure of an activated DNA-PK and its implications for NHEJ.

• DOI: 10.1016/j.molcel.2020.12.015
• 发表时间: 2021-02-18
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Chen X;Xu X;Chen Y;Cheung JC;Wang H;Jiang J;de Val N;Fox T;Gellert M;Yang W
• 通讯作者: Yang W

An activation-induced cytidine deaminase-independent mechanism of secondary VH gene rearrangement in preimmune human B cells.

免疫前人类 B 细胞中二次 VH 基因重排的激活诱导的不依赖胞苷脱氨酶的机制。

• DOI: 10.4049/jimmunol.181.11.7825
• 发表时间: 2008
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Longo,NancyS;Grundy,GabrielleJ;Lee,Jisoo;Gellert,Martin;Lipsky,PeterE
• 通讯作者: Lipsky,PeterE

Role of RAG1 autoubiquitination in V(D)J recombination.

RAG1 自动泛素化在 V(D)J 重组中的作用。

• DOI: 10.1073/pnas.1510464112
• 发表时间: 2015
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Singh,SamarendraK;Gellert,Martin
• 通讯作者: Gellert,Martin

Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.

• DOI: 10.1016/j.molcel.2009.06.022
• 发表时间: 2009-07-31
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Grundy, Gabrielle J.;Ramon-Maiques, Santiago;Dimitriadis, Emilios K.;Kotova, Svetlana;Biertuempfel, Christian;Heymann, J. Bernard;Steven, Alasdair C.;Gellert, Martin;Yang, Wei
• 通讯作者: Yang, Wei

Crystal structure of the V(D)J recombinase RAG1-RAG2.

• DOI: 10.1038/nature14174
• 发表时间: 2015-02-26
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Kim, Min-Sung;Lapkouski, Mikalai;Yang, Wei;Gellert, Martin
• 通讯作者: Gellert, Martin