Molecular Basis of Immunoglobulin Heavy Chain Switch

描述（由申请人提供）：抗体（免疫球蛋白，Ig）类切换使B淋巴细胞从产生IgM切换到产生IgG、IgA或IgE，从而提高抗体清除体内病原体和细菌毒素的能力。类转换发生在染色体内DNA重组事件，必须仔细控制，以避免与其他染色体的异常重组（易位）。然而，癌基因和IgH位点之间确实发生易位，这可能导致B细胞淋巴瘤。在类别转换过程中，激活诱导胞苷脱氨酶（AID）在Ig重链基因位点（IgH）的开关(S)区域启动DNA双链断裂（DSBs）的形成，这是类别转换所必需的。我的第一个目标是确定AID如何在Ig S区域分解dC，形成dU，从而导致dsb。我们已经证明，艾滋病诱导的dC脱胺通过碱基切除修复途径导致DNA单链断裂（SSBs）。这些ssb如何转化为dsb就不太清楚了。我们已经报道了另一种DNA修复途径，错配修复（MMR）在这一步骤中很重要，我们将研究它的作用。我们将通过确定AID诱导的S区dU的频率和位置来研究ssb如何转化为dsb， AID靶点的频率和位置如何影响转换频率，以及MMR蛋白是否可能被AID自身招募到S区。在Aim 2中，我们将在本资助的当前期限内继续我们的发现，即AID可以在活化的B细胞中的IgH位点以外的位置激发dsb。我们将确定是什么使这些其他位点成为AID的靶点，以及这些dsb是否导致染色体断裂、缺失和易位，以及MMR和其他已知参与CSR的DNA修复蛋白（例如ATM、H2AX和53BP1）是否参与制造或阻止这些dsb。

DESCRIPTION (provided by applicant): Antibody (immunoglobulin, Ig) class switch causes B lymphocytes to switch from producing IgM to producing IgG, IgA or IgE, which improves the ability of the antibody to remove pathogens and bacterial toxins from the body. Class switching occurs by an intrachromosomal DNA recombination event that must be carefully controlled in order to avoid aberrant recombination with other chromosomes (translocations). However, translocations do occur between oncogenes and the IgH locus, and this can lead to B cell lymphomas. During class switching, activation-induced cytidine deaminase (AID) initiates the formation of DNA double strand breaks (DSBs) at switch (S) regions in the Ig heavy chain gene locus (IgH), which are necessary for class switching. My first Aim is to determine how deamination of dC's in Ig S regions by AID, forming dU's, results in DSBs. We have shown that AID-induced deamination of dC leads to DNA single-strand breaks (SSBs) via the base excision repair pathway. How these SSBs are then converted to DSBs is less clear. We have reported that another DNA repair pathway, mismatch repair (MMR) is important for this step, and we will investigate its role. We will investigate how SSBs are converted to DSBs by determining the frequency and sites of AID- induced dU's in S regions, how the frequency and positions of AID targets affects frequency of switching, and whether MMR proteins might be recruited to S regions by AID itself. In Aim 2 we will follow up on our finding during the current term of this grant that AID can instigate DSBs at sites other than the IgH locus in activated B cells. We will determine what makes these other sites targets for AID, and if these DSBs lead to chromosome breaks, deletions and translocations, and whether MMR and other DNA repair proteins known to be involved in CSR, for example, ATM, H2AX, and 53BP1 are involved in making or preventing these DSBs. PUBLIC HEALTH RELEVANCE: The majority of human mature B-cell lymphomas are characterized by reciprocal chromosomal translocations that often involve the immunoglobulin (Ig) genes. The longstanding assumption that these translocations are generated by erroneous antibody class switching, a normal process that is essential for an effective humoral immune response, was verified only recently. Antibody class switching is a process that requires the introduction of DNA breaks into the Ig genes, into special regions called switch regions, and then joining one of these DNA break ends with another DNA break end in the same Ig locus, on the same chromosome. The mechanism of induction of DNA breaks and also how this process is regulated to prevent joining the DNA breaks to another chromosome, which can result in activation of oncogenes and thus B cell lymphomas not understood. The experiments proposed here will determine how the activity of the enzyme AID, which initiates class switching and also chromosomal translocations, is regulated and how it chooses its targets. We have shown that a specific DNA repair pathway in cells, called mismatch repair, which is essential for repairing mistakes during DNA synthesis, is required for formation of most of the DNA breaks during class switching. We seek to further understand its role in converting AID-induced lesions to DNA breaks. Mismatch repair is an essential repair system for eliminating mutations and microsatellite instability, thereby protecting against cellular transformation and malignancy. The hypothesis that this pathway increases DNA breaks seems counterintuitive, but arises directly from our understanding of the role of this pathway during class switching. AID is required for chromosomal translocations between the c-myc oncogene and the Ig locus in mature B-cell plasmacytomas in mouse. Also, many human B-cell lymphomas that originate from germinal center B cells show chromosomal translocations involving the Ig switch regions, suggesting the involvement of class switch processes. IgH translocations often greatly increase oncogene expression due to juxtaposition to the strong IgH enhancers, leading to cellular transformation and malignancy. Whether AID targeting to other loci besides the Ig loci leads to formation of DNA breaks at these sites is, however, unknown. By the use of a genome- wide search method, we have found AID-dependent DNA breaks at several other sites in the genome. We will examine whether these other sites are involved in chromosomal translocations with the Ig locus. We will investigate what makes these other sites targets for AID, and for DNA break formation. Thus our studies will also shed light on the important question of how AID chooses its targets.