Patricia Cortes - US grants

nucleic acid sequence; DNA repair; severe combined immunodeficiency; green fluorescent proteins; DNA damage; biochemistry; western blottings; transfection; polymerase chain reaction; flow cytometry;

During lymphocyte development, antigen receptor genes are assembled from germline V,D and J gene segments by a site-specific somatic recombination process termed as V(D)J recombination. Defects in this pathway result in a number of primary immunodeficiencies like Severe Combined Immuno Deficiency (SCID) and Omenn syndrome and potentially can lead to development of leukemias and lymphomas. Two lymphoid specific proteins RAG1 and RAG2 cleave DNA between the coding segments and signal sequences. The Non-Homologous End Joining (NHEJ) DNA repair pathway repairs the double strand breaks thus generated. This reaction results in coding ends with covalently closed hairpin structures that need to be opened and processed before ligation. Mutations in a novel protein called Artemis causes RS-SCID syndrome, a SCID phenotype associated with radiosensitivity. Data from in vitro analysis and knockout mice have shown that Artemis is the nuclease that opens hairpin-coding ends. However, nothing is known about the biochemistry and structure of this protein. Further, direct evidence for its role in NHEJ is still lacking. Also, the C-terminal region of Artemis that forms almost half of the protein has not been studied. In this grant application, we describe a series of experiments that will address these issues while extending our observation that Artemis interacts with Ligase IV/XRCC4 in vivo through its C-terminal domain and this complex is very active in DSB ligation in vitro. Through specific aims proposed we will 1) characterize the biochemical properties of Artemis and develop its crystal structure, 2) investigate the in vivo relevance of the interaction of Artemis with Ligase IV in NHEJ and define a role of its C-terminal region in NHEJ and 3) establish the significance of this interaction in V(D)J recombination and ascertain the role of its C-terminal region. Together, the experiments proposed for the specific aims will begin to define the structure-function relationship of Artemis and the mechanism of different RS-SCID mutations found in humans. Further, by establishing the importance of interaction of Artemis with Ligase IV in both V(D)J recombination and NHEJ in vivo, we will provide novel insights into multiple roles played by Artemis in these pathways. Through this study, we will also describe a novel protein structure, by solving the protein structure of Artemis and further define its role in development of a healthy immune system.

During development of the immune system, antigen receptor genes in B and T cells are assembled from germline coding gene segments by a site-specific somatic recombination process termed V(D)J recombination. Defects in this pathway result in a number of primary immunodeficiencies such as Severe Combined Immuno Deficiency (SCID) and Omenn Syndrome and have the potential to result in development of leukemias and lymphomas. To prevent development of such diseases, there is a need to properly resolve the DNA intermediates generated during the recombination reaction. These include DNA double strand breaks (DSB) that must be repaired by the NHEJ pathway, and DNA hairpin structures that must be opened and processed during V(D)J recombination. Mutations in a novel protein called Artemis causes RS-SCID syndrome, a SCID phenotype associated with radiosensitivity. Data from in vitro analysis and knockout mice have shown that Artemis is the nuclease that opens hairpin-coding ends during V(D)J recombination. However, little is known about the biochemistry and structure of Artemis. Further, direct evidence for its role in NHEJ is still lacking. Furthermore, the C-terminal region of Artemis, which constitutes almost half of the protein, remains poorly characterized and the contribution of Artemis- endonucleasedefective mutants to V(D)J recombination has also not been analyzed. In this grant application, we describe a series of experiments that will address these issues while extending observations we have regarding multiple and yet uncharacterized roles of Artemis in V(D)J recombination and DNA repair. Through specific aims proposed we will characterize the biochemical properties of Artemis (oligomerization, DNA binding and nuclease activity) and develop its crystal structure (aim 1), investigate the in vivo relevance of novel protein interactions and multiple functions of Artemis in NHEJ (aim2) and V(D)J recombination (aim 3). Together, the experiments proposed in the specific aims will begin to define the structure-function relationship of Artemis and the mechanism of different RS-SCID mutations found in humans. Further, by establishing the importance of novel protein interactions of Artemis and its multiple functions in both V(D)J recombination and NHEJ, we will provide new insights into the molecular mechanism of these processes. Through this study, we will also describe the protein structure of Artemis and further define its role(s) in development of a healthy immune system.

DESCRIPTION (provided by applicant): Ligase IV has been shown to play critical roles in Non-Homologous End Joining (NHEJ), V(D)J recombination and development of the immune system, stem cell exhaustion, ageing and neural growth and development, as exemplified by the phenotype of patients with hypomorphic mutations in Ligase IV. Numerous proteins have been shown to modulate Ligase IV activity. Among them, XRCC4 and Cernunnos/XLF are perhaps the best/most conclusively characterized. Both of these factors, like Ligase IV, are part of the core-NHEJ machinery. Human mutations in XRCC4 have not been described yet, but interestingly, Cernunnos/XLF mutations in humans result in growth defects, microcephaly and immunodeficiency, similar to what has been observed for Ligase IV mutations. Our recent findings have identified Artemis, a factor also linked to immunodeficiency in humans, as a protein that directly interacts with Ligase IV, in addition we have observed regulation of XRCC4 by Ligase IV. In this project we propose to analyze the functional relevance of these novel Ligase IV/Artemis complex in genomic stability and V(D)J recombination. The mechanism by which Ligase IV regulates XRCC4 function, and the composition and function of endogenous Ligase IV complexes in lymphocytes will also be investigated. Our preliminary work with Ligase IV recombinant protein and protein fragments has resulted in crystallization of a Ligase IV fragment containing the Ligase IV DNA binding domain. Experiments are proposed to solve the structure of Ligase IV and/or Ligase IV in complex with Artemis. All these are challenging yet very relevant experiments for which we show significant progresses. Furthermore, the impact of mutations identified in patients with LIG4 syndrome on regulation of its function by Artemis and on how these mutant Ligase IV proteins affect XRCC4 function will also be investigated. Information gained from the proposed studies will contribute to a molecular and structural understanding of NHEJ, V(D)J recombination and human diseases linked to defects in both of these processes. PUBLIC HEALTH RELEVANCE: Despite important progress in our understanding of Ligase IV function, its structure remains largely uncharacterized and our preliminary data suggest novel mechanisms of regulation that remain to be unraveled. This project proposes to investigate our preliminary finding on novel Ligase IV interactions and functions by using in vivo and in vitro approaches;we also propose studies to solve the structure of Ligase IV and Ligase IV complexes. By increasing our knowledge of Ligase IV structure, function and regulation, in V(D)J recombination and DNA repair, the proposed work will contribute to our understanding of human disease, specifically, Ligase IV and Artemis mediated, immunodeficiency and cancer and will facilitate the path to find better treatments for this debilitating diseases.

DESCRIPTION (provided by applicant): V(D)J recombination is a site-specific somatic recombination reaction whose end products are genes encoding cell surface receptors and antibody molecules that are central to immune system function. Proper recombination is absolutely required for development of a healthy immune system: when the reaction does not occur or is poorly controlled, there is the potential for development of immunodeficiencies, leukemias, and lymphomas. Our long-term goal is to understand the mechanisms of V(D)J recombination and its regulation, and our approach has been through a biochemical analysis of the RAG proteins. Though much progress has been made in the field of recombination in the last two decades, there are still numerous questions outstanding, among them are the issue of accessibility and the regulation of recombination in vivo. In this grant application, we describe a series of experiments that will address these issues while extending our findings that showed interaction of Rag2 with core histones and the MCM complex. Through specific aims proposed, we will: 1) Investigate the requirement and participation of MCM2-7 complex and MCM associated proteins in V(D)J recombination;2) Study the role of Rag2 C-terminus, acidic region and PHD domain, in the different steps of V(D)J recombination as well as their participation in the various protein-protein interactions mediated by this region;3) Analyze the RSS cleavage mechanisms that depend on Rag2 full-length using a chromatin substrate. The focus of these aims is to understand the molecular mechanism that determine regulation of V(D)J recombination by Rag2 and specifically the contribution of Rag2 C-terminus to this process. PUBLIC HEALTH RELEVANCE: V(D)J recombination is an essential process that takes place in B and T lymphocytes. This process is mediated by recombination factors that play unique and fundamental roles;deregulation of any of the required factors results in severe immunodeficiency. This project is focused on understanding how Rag2, one of the recombination factors, regulated the reaction by mediating multiple protein interactions and thus modulating the generation of a diverse immune system.