MECHANISM OF DNA RECOMBINATION AT CLASS SWITCH SEQUENCES

类别转换序列的 DNA 重组机制

• 批准号: 7330348
• 负责人: MICHAEL R LIEBER
• 金额: $ 26.27万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7330348
• 关键词: Affect; Autoimmunity; B-Lymphocytes; Back; Chromosomes; Code; DNA; DNA ligase IV; DNA-Directed RNA Polymerase; Dependence; Enzymes; Funding; Gene Rearrangement; Genetic Recombination; Genetic Transcription; Hybrids; Hydrogen Bonding; Hypersensitivity; IgE; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Switch Recombination; Immunologics; In Vitro; Infection; Length; Location; Malignant Neoplasms; Movement; Pathway interactions; Process; RNA; RNA Splicing; Relative (related person); Repetitive Sequence; Site; Structure; Substrate Specificity; Testing; Transcript; Work; activation-induced cytidine deaminase; enzyme activity; in vivo; mutant; neoplastic

Class switch recombination (CSR) is the somatic DNA rearrangement process by which B lymphocytes change their heavy chains from IgM to IgG, IgA or IgE. This DNA recombination process is distinctive among all eukaryotic (and prokaryotic) recombination mechanisms because it is the only one in which the recombination occurs within long repetitive regions in a manner that is dependent on transcription through the region such that a G-rich transcript is generated. Understanding of the mechanism of this process is important for immunologic reasons (infection, allergy, autoimmunity) and for its cancer-relevance (neoplastic translocations that occur within the class switch sequences, such as the t(8;14)). In the previous funding cycle, we showed that class switch regions assume a distinctive structure within the chromosomes of appropriately stimulated B cells. This structure is an R-loop in which the G-rich (nontemplate) strand of the repetitive switch regions is single-stranded for lengths exceeding 1 kb. The G-rich RNA that is generated by transcription through the switch region remains annealed to the C-rich (template) strand of the switch region. These results explain the in vivo orientation-dependence of the transcription requirement, which we documented in our earlier switch recombination work. In the current proposal, Aim 1 examines whether the chromosomal R-loop is limited to the switch repeats and whether it is affected by splicing. Aim 2 examines the mechanism of R-loop formation, and this is optimally done in vitro where we probe the structure with enzymes; study the repeat length and sequences needed for R-loop formation; vary the rate of RNA polymerase movement; and vary the number of hybrogen bonds in the R-loop. Aim 3 examines the interface between AID enzyme activity and R-loops. Can purified AID deaminate both DNA strands in the R-loop? Is the proximity of WRC sequence motifs on the two DNA strands critical for double-strand break formation? Aim 4 tests whether DNA ligase IV is required for CSR. This aim is directed at providing some clarification of what end joining pathway is used for class switch recombination. Hence, this proposal tests in vivo substrate structure, its mechanism of formation, the action of AID enzyme on that structure, and the pathway for rejoining the ends after breakage has occurred.

类开关重组（CSR）是b 淋巴细胞将其重链从IgM变为IgG，IgA或IgE。此DNA重组过程是 在所有真核（和原核生物）重组机制中，它是独特的，因为它是唯一一种 重组以依赖的方式发生在长度重复区域内 通过该区域的转录，从而产生了富含G的转录本。理解 由于免疫原因（感染，过敏，自身免疫性），此过程的机制很重要 它的癌症相关性（在类开关序列中发生的肿瘤易位，例如 t（8; 14））。在上一个融资周期中，我们表明班级开关区域假设一个独特的结构 在适当刺激的B细胞的染色体内。该结构是R环，其中G富含G （非板）重复开关区域的链，长度超过1 kb。这 通过转录通过开关区域产生的富含G富含G的RNA仍然将其退火到富含C （模板）开关区域的链。这些结果解释了体内方向的依赖性 转录需求，我们在早期的开关重组工作中记录了这一点。在电流中 提案，AIM 1检查染色体R-loop是否仅限于开关重复以及它是否限于 受剪接影响。 AIM 2检查了R环形成的机制，这是最佳的 我们用酶探测结构的体外；研究重复长度和序列 R环形成；改变RNA聚合酶运动的速率；并改变杂种键的数量 R环。 AIM 3检查了AID酶活性和R-loops之间的接口。可以纯化的援助 脱氨酸在R环中的两个DNA链？是两个DNA上WRC序列基序的接近度 双链断裂形成至关重要的链？ AIM 4测试CSR是否需要DNA连接酶IV。 此目标旨在提供一些澄清的连接途径用于类开关 重组。因此，该建议在体内底物结构中测试了其形成机理， 援助酶对该结构的作用，以及断裂后重新加入末端的途径 发生。