Mechanism of Immunoglobulin Hypermutation

描述（由申请人提供）：免疫球蛋白（Ig）基因的体细胞超突变（SHM）过程由胞苷脱氨酶AID启动，产生胞苷(C)到尿苷(U)的过渡突变。在SHM外，U通过碱基切除修复和DNA错配修复忠实地修复为C。在SHM期间，矛盾的是，这些修复机制与翻译DNA聚合酶一起被招募，通过容易出错的修复在U和U附近产生突变。这项拨款申请旨在研究与SHM过程有关的两个主要问题。首先，突变是如何被限制在离启动子约2kb的地方，而保留了转录的5< 100bp的Ig基因？我们提出了一个模型，其中AID装载在启动子附近的转录复合体上，与RNA聚合酶一起移动，并随机地从复合体中分离，同时在其轨道上脱氨胞嘧啶。一旦分离，AID就不能重新加载到转录机制上。另一种模型是，AID被松散地吸引到Ig基因座上（由于我们最近发现的与Ig增强子中的元件结合的必不可少的顺式元件），但不随转录复合物移动。我们计划在SHM期间确定AID和RNA聚合酶在AID靶基因上的位置。我们还将跟进初步发现，即艾滋病确实需要转录过程本身。我们将进一步研究核小体的作用，核小体似乎是SHM的屏障。这些实验对于了解SHM如何产生可变区突变而Ig基因的恒定区不受突变的影响非常重要，这些事实分别对增强和保护可变区和恒定区的防御和生理作用至关重要。另一个问题与其他人和我们实验室的新发现有关，即在非Ig基因和Ig基因中，有相当大比例的尿嘧啶是由AID产生的，并且许多艾滋病诱导的尿嘧啶可以在两种类型的基因中无错误地修复，而另一部分则以容易出错的方式处理。我们已经证明，在Ig基因和非Ig基因中，AID病变的无错误修复确实发生。我们建议制造表达修饰的AID分子的突变B细胞系，这些修饰的AID分子要么只在G1期表达，要么只在S、G2和m期表达。在细胞周期中，AID表达的时间将影响其他蛋白质与AID和艾滋病诱导的尿嘧啶相互作用，从而决定尿嘧啶及其邻近序列是发生突变还是被无错误修复。大多数实验将首先在经过体细胞超突变的培养B细胞系中进行。如果在培养细胞中得出结论，将在正常小鼠和转基因小鼠中进行一些实验。

DESCRIPTION (provided by applicant): The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes is initiated by the cytidine deaminase AID creating cytidine (C) to uridine (U) transition mutations. Outside of SHM, U is faithfully repaired to C with the help of base excision repair and DNA mismatch repair. During SHM, paradoxically, these repair mechanisms are recruited together with translesion DNA polymerases to create mutations at and near the U by error-prone repair. This grant application proposes to study two major questions concerning the SHM process. One, how are the mutations restricted to about 2 kb from the promoter and sparing the transcribed 5< 100 bp of the Ig genes? We have proposed a model where AID loads on the transcription complex near the promoter, travels with the RNA polymerase, and stochastically dissociates from the complex while deaminating cytosines in its track. Once dissociated, AID cannot re-load onto the transcription machinery. An alternative model is that AID is attracted loosely to Ig loci (due to cis-elements that we have recently identified as essential, in combination with elements in the Ig enhancers) but does not travel with transcription complexes. We plan to determine the location of AID and RNA polymerase on the AID target gene during SHM. We will also follow up on preliminary findings that AID indeed requires the transcription process itself. We will further investigate the role of nucleosomes that appear to be a barrier to SHM. These experiments will be important to learn how SHM can create variable region mutations whereas the constant region of Ig genes is spared from mutations, facts that are essential to enhance and protect the defensive and physiological roles of the variable and constant regions, respectively. The other question relates to new findings by others and our lab that a considerable proportion of uracils are created by AID in non-Ig genes as well as in Ig genes and that many of the AID-induced uracils can be repaired error-free in both types of genes, while another proportion is treated in an error-prone fashion. We have shown, that error-free repair of AID lesions does occur in Ig genes as well as in non-Ig genes. We propose to produce mutating B cell lines that express modified AID molecules that are either expressed only during the G1- phase or only during S, G2, and M. The time in the cell cycle when AID is expressed will affect which other proteins interact with AID and with the AID-induced uracil, thus determining whether the uracil and neighboring sequences become mutated or are repaired error-free. Most of the experiments will at first be carried out in B cell lines in culture that undergo somatic hypermutation. If conclusive in cultured cells, some experiments will be checked in normal and transgenic mice.