Function of the AID C terminus in Ig class switching

描述（由申请人提供）：这是一项探索性资助申请，旨在建立我们的初步结果，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）期间的重组步骤很重要。几年来已经知道AID的C末端10个氨基酸对于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，该过程也依赖于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是重要的。我们已经获得了新的结果，表明在诱导进行CSR的脾B细胞中，AID与参与将DNA断裂引入S区的其他酶（特别是UNG和Msh 2-Msh 6）协同结合到IG开关（S）区，并且这种结合依赖于AID C末端。利用逆转录病毒在AID的AID-/-小鼠脾B细胞中的过表达，我们检测了AID与S？而S？在染色质免疫沉淀（ChIP）试验中，而C末端缺失的AID（？AID）不与S区DNA进行ChIP。同样，UNG和Msh 2-Msh 6也被ChIP在S？在Aid-/-细胞表达中的作用AID，但在表达AID的细胞中未检测到它们，这表明这些修复蛋白的结合依赖于AID C末端，并且AID和这些蛋白的结合可能是协同的，即共依赖的。与这些蛋白质结合协同S？DNA，在ung-/-aid-/- B细胞或msh 2-/-aid-/- B细胞中，转导的完全脱辅基AID不能在S？通过Chip这些结果表明，为了使AID和UNG以及Msh 2-Msh 6在S区充分稳定地结合以被ChIP检测到，它们必须彼此协同结合DNA，并且这种协同结合依赖于AID C末端。我们提出验证以下假设：（1）AID C末端通过中间蛋白将UNG和Msh 2-Msh 6募集到S区，以及（2）AID的C末端对于在细胞周期的G1期将UNG和Msh 2-Msh 6募集到S区是重要的，和（3）AID的C末端对于随后形成DNA断裂的步骤是重要的，所述DNA断裂将CSR导向非同源末端连接（NHEJ）。为了验证这些假设，我们提出了3个具体的目的：1）研究AID与UNG和Msh 2-Msh 6之间的相互作用机制。2)确定AID募集UNG和Msh 2-Msh 6是否对G1期S区DSB的产生以及G1期DSB的修复重要。在正常脾B细胞，艾滋病依赖性S？DSB仅限于G1阶段。然而，在其他细胞类型中，UNG和Msh 2-Msh 6在S期期间被DNA复制复合物募集到DNA。3)确定AID C末端是否对将参与NHEJ的酶募集到S区并由此将CSR导向NHEJ重要。

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.