Alternative End-Joining in DNA Repair and Chromosomal Translocations

DNA 修复和染色体易位中的选择性末端连接

• 批准号: 9099801
• 负责人: Vipul Kumar
• 金额: $ 3.16万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9099801
• 关键词: Antibodies; Antigen Receptors; B-Cell Lymphomas; B-Lymphocytes; BCL2 gene; Biological Assay; CRISPR/Cas technology; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cell division; Cells; Chemicals; Chimeric Proteins; Chromosomal Breaks; Chromosomal Rearrangement; Chromosomal translocation; Chromosome Deletion; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytogenetics; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence Rearrangement; Data; Detection; Double Strand Break Repair; Embryo; Exons; G1 Arrest; G22P1 gene; Gene Mutation; Gene Silencing; Generations; Genome; Genome Stability; Glucocorticoid Receptor; Health; Heavy-Chain Immunoglobulins; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; J segment gene; Lead; Ligation; Lymphocyte Activation; Lymphomagenesis; Malignant Neoplasms; Mature B-Lymphocyte; Mediating; Mus; Mutant Strains Mice; Nonhomologous DNA End Joining; Oncogenes; Oncogenic; Pathway interactions; Phase; Phosphotransferases; Poly(ADP-ribose) Polymerases; Proteins; Publishing; Reporter; Role; Site; Southern Blotting; System; Testing; Transgenes; V(D)J Recombination; XRCC1 gene; XRCC4 gene; XRCC5 gene; abl Oncogene; activation-induced cytidine deaminase; ataxia telangiectasia mutated protein; base; endodeoxyribonuclease SceI; endonuclease; genome-wide; homologous recombination; insight; interest; nuclease; p53-binding protein 1; repaired; research study; sensor

DESCRIPTION (provided by applicant): Improperly repaired DNA double-strand breaks (DSBs) can lead to cell death or chromosomal rearrangements that contribute to oncogenic transformation. The two major mammalian DSB repair pathways are homologous recombination (HR), which is active in post-replicative (S/G2) cells, and classical non-homologous end joining (C-NHEJ), which predominates in pre-replicative (G1) cells. For C-NHEJ, the Ku70/Ku80 heterodimer ("Ku") recognizes DSBs and the XRCC4/Ligase4 (Lig4) complex joins them. During V(D)J recombination in developing B lymphocytes, RAG endonuclease generated DSBs at V, D, and J gene segments are joined exclusively by C-NHEJ to assemble exons encoding antigen receptor/antibody variable regions. During immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes, activation-induced cytidine deaminase (AID)-initiated DSBs in IgH switch (S) regions are fused, predominantly by C-NHEJ, to exchange expressed IgH constant region exons. Aberrant joining of V(D)J- or CSR-associated DSBs can lead to chromosomal translocations that fuse antigen receptor loci to oncogenes, and, thereby, contribute to lymphomagenesis. In the absence of C-NHEJ, RAG- or AID-initiated IgH DSBs can be joined to form such oncogenic chromosomal translocations by an alternative end-joining (A-EJ) pathway (or pathways). In addition, CSR is carried out relatively robustly by A-EJ in the absence of Lig4, Ku, or both. Based on substantial preliminary data, we propose the A-EJ in the absence of Lig4 is distinct from that which occurs in the absence of Ku (or Ku plus Lig4). We refer to these two A-EJ pathways as "Lig4-independent" and "Ku- independent" A-EJ, respectively. We propose to elucidate DSB recognition and joining components of the two A-EJ pathways, their relative activity in the G1 cell cycle, and their relative contributions to normal versus aberrant end-joining that promotes chromosomal translocations. Specifically, we propose to 1) Identify factors that mediate Lig4- and/or Ku-independent A-EJ during CSR and 2) Assess potential roles of Lig4- and/or Ku- independent A-EJ in G1-arrested pro-B lines.

描述(由申请人提供)：修复不当的DNA双链断裂(DSB)可导致细胞死亡或染色体重排，从而导致致癌转化。哺乳动物DSB修复的两条主要途径是在复制后(S/G2)细胞中活跃的同源重组(HR)和在复制前(G1)细胞中占优势的经典非同源末端连接(C-NHEJ)。对于C-NHEJ，Ku70/Ku80杂二聚体(Ku)识别DSB，XRCC4/Ligase4(Lig4)复合体加入它们。在发育中的B淋巴细胞的V(D)J重组过程中，V、D和J基因片段上RAG内切酶产生的DSB被C-NHEJ独占地连接在一起，组装编码抗原受体/抗体可变区的外显子。在激活的成熟B淋巴细胞的免疫球蛋白重链(Ig)重链(IgH)类开关重组(CSR)过程中，激活诱导的胞苷脱氨酶(AID)启动的IgH开关(S)区域的DSB被融合，主要由C-NHEJ融合，以交换表达的IgH恒定区外显子。V(D)J或CSR相关DSB的异常连接可导致染色体易位，将抗原受体基因与癌基因融合，从而促进淋巴肿大。在没有C-NHEJ的情况下，RAG或AID启动的IgH DSB可以通过另一条末端连接(A-EJ)途径连接起来，形成这种致癌的染色体易位。此外，在没有Lig4和/或Ku的情况下，A-EJ执行CSR的力度相对较大。基于大量的初步数据，我们认为没有Lig4的A-EJ与没有Ku(或Ku+Lig4)的A-EJ是不同的。我们将这两条A-EJ途径分别称为“Lig4非依赖性”和“Ku非依赖性”A-EJ。我们建议阐明两条A-EJ通路的DSB识别和连接成分，它们在G1细胞周期中的相对活性，以及它们在促进染色体易位的正常和异常末端连接中的相对贡献。具体地说，我们建议1)确定在CSR过程中介导Lig4和/或Ku非依赖性A-EJ的因素，以及2)评估Lig4和/或Ku无关的A-EJ在G1受阻的Pro-B系中的潜在作用。