Molecular Basis of Immunoglobulin Heavy Chain Switch

描述（由申请方提供）：抗体（免疫球蛋白，IG）类别转换导致B淋巴细胞从产生IgM转为产生IgG、伊加或IgE，从而提高抗体清除体内病原体和细菌毒素的能力。类别转换通过染色体内DNA重组事件发生，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，这可导致B细胞淋巴瘤。在类别转换期间，激活诱导的胞苷脱氨酶（AID）在IG重链基因座（IgH）中的开关（S）区域启动DNA双链断裂（DSB）的形成，这是类别转换所必需的。我的第一个目标是确定AID如何使IG S区的dC脱氨基，形成dU，导致DSB。我们已经表明，艾滋病诱导的dC脱氨基导致DNA单链断裂（SSB）通过碱基切除修复途径。这些SSB随后如何转换为DSB尚不清楚。我们已经报道了另一种DNA修复途径，错配修复（MMR）对这一步很重要，我们将研究它的作用。我们将通过确定S区域中AID诱导的dU的频率和位点，AID靶标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本身募集到S区域来研究SSB如何转化为DSB。在目标2中，我们将在本基金的当前期限内继续研究我们的发现，即AID可以在活化的B细胞中IgH位点以外的位点引发DSB。我们将确定是什么使这些其他位点成为AID的靶点，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）是否参与制造或预防这些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.