Function of the AID C terminus in Ig class switching

描述(由申请人提供)：这是一项探索性拨款的申请，以我们的初步结果为基础，该结果表明，激活诱导的胞苷脱氨酶(AID)的C末端对于抗体类别转换重组(CSR)中的重组步骤是重要的。已知AID的C端10个氨基酸对CSR非常重要，尽管它们在抗体基因的体细胞超突变(SHM)过程中似乎没有任何作用，这一过程也依赖于AID。此外，AID C末端对于防止IgH和c-myc基因座之间的染色体易位也很重要。我们的新结果表明，在被诱导为CSR的脾B细胞中，AID与参与将DNA断裂引入S区域的其他酶协同结合到Ig开关(S)区域，特别是UNG和Msh2-MSH6，并且这种结合依赖于AID C末端。利用逆转录病毒在艾滋病小鼠脾B细胞中的过度表达，检测艾滋病病毒与S的结合。S呢？？在染色质免疫沉淀(CHIP)检测中，C端缺失的AID(？AID)不与S区域DNA发生芯片反应。同样，在S的芯片中也检测到了UNG和Msh2-MSH6。但在表达AID的细胞中未检测到，提示这些修复蛋白的结合依赖于AID的C末端，AID与这些蛋白的结合可能是协同的，即相互依赖的。与这些蛋白质与S协同结合的假设一致吗？S未检测到转导全长AID的Un-/-Aid-/-B细胞或MsH2-/-Aid-/-B细胞中的DNA？用芯片。这些结果表明，Aid、UNG和Msh2-MSH6要足够稳定地结合到S区域的芯片上，它们必须相互协同结合DNA，这种协同结合依赖于AID C末端。我们建议检验假设(1)AID的C末端通过中间蛋白将UNG和Msh2-MSH6招募到S区域，(2)在细胞周期的G1期，AID的C末端对于将UNG和Msh2-MSH6招募到S区域是重要的，(3)AID的C末端对于DNA断裂的形成是重要的，DNA断裂将CSR导向非同源末端连接(NHEJ)。为了验证这些假说，我们提出了三个具体目标：1)研究AID与UNG和Msh2-MSH6的相互作用机制。2)研究AID对UNG和Msh2-MSH6的募集是否对S区G1期双链断裂的产生和G1期双链断裂的修复起重要作用。在正常脾B细胞中，艾滋病依赖的S？双链断裂被限制在G1相。然而，在其他类型的细胞中，UNG和Msh2-MSH6在S期被DNA复制复合体招募到DNA中。3)确定AID C末端是否对将NHEJ相关酶招募到S区域，从而将CSR导向NHEJ起重要作用。

DESCRIPTION (provided by applicant): This is an application for an exploratory grant to build on our preliminary results suggesting that the C terminus of activation-induced cytidine deaminase (AID) is important for the recombination step during antibody class switch recombination (CSR). It has been known for several years that the C terminal 10 amino acids of AID are very important for CSR, although they do not appear to have any role during somatic hypermutation (SHM) of antibody genes, a process also dependent upon AID. Also, the AID C terminus is important for preventing chromosomal translocations between the IgH and c-myc loci. We have obtained novel results indicating that in splenic B cells induced to undergo CSR, AID binds to Ig switch (S) regions cooperatively with other enzymes involved in introducing DNA breaks into S regions, specifically UNG and Msh2-Msh6, and that this binding is dependent upon the AID C terminus. Using retroviral over-expression in aid-/- mouse splenic B cells of AID, we detect AID binding to S? and S?? in chromatin immunoprecipitation (ChIP) assays, whereas C terminal deleted AID (?AID) does not ChIP with S region DNA. Likewise, both UNG and Msh2-Msh6 are also detected by ChIP at S? in aid-/- cells expressing ?AID, but they are not detected in cells expressing AID, suggesting that the binding of these repair proteins depends on the AID C terminus, and the binding of AID and these proteins might be cooperative, i.e. co-dependent. Consistent with the hypothesis that these proteins bind cooperatively to S? DNA, in ung-/-aid-/- B cells or in msh2-/-aid-/- B cells, transduced full lengt AID cannot be detected at S? by ChIP. These results suggest that in order for AID and UNG and Msh2-Msh6 to bind sufficiently stably to be detected by ChIP at S regions, they must bind DNA cooperatively with each other, and this cooperative binding depends on the AID C terminus. We propose to test the hypotheses (1) that the AID C terminus recruits UNG and Msh2-Msh6 to S regions via an intermediary protein, and (2) that the C terminus of AID is important for recruiting UNG and Msh2-Msh6 to S regions during the G1 phase of the cell cycle, and (3) that the C terminus of AID is important for steps subsequent to formation of DNA breaks that direct CSR towards non-homologous end-joining (NHEJ). In order to test these hypotheses we propose 3 Specific Aims: 1) To investigate the mechanism of interaction between AID and UNG and Msh2-Msh6. 2) To determine if the recruitment of UNG and Msh2-Msh6 by AID is important for creating DSBs in S regions during G1 phase and also for their repair during G1 phase. In normal splenic B cells, AID-dependent S? DSBs are restricted to the G1 phase. However, in other cell types, UNG and Msh2- Msh6 are recruited by the DNA replication complex to DNA during S phase. 3) To determine if the AID C terminus is important for recruiting enzymes involved in NHEJ to S regions and thereby directing CSR toward NHEJ.