Activation-induced Deaminase and Antibody Maturation

描述（由申请人提供）：活化诱导脱氨酶（AID）属于APOBEC家族酶，其将核酸中的胞嘧啶转化为尿嘧啶。AID是抗体成熟所必需的两个过程-体细胞超突变和类别转换重组所必需的。此外，AID还已知会引起癌基因的超突变并促进染色体易位，这是癌症的标志。关于AID在致癌作用中的作用的未回答的问题之一是AID如何选择某些基因和某些位点进行脱氨基作用。在上一个资助期间，我们研究了转录在靶向过程中所起的作用;在本提案中，我们将研究这种酶的底物选择如何影响遗传结果。我们将确定当AID的底物特异性改变时，遗传改变的频率、位置和谱如何改变。为了实现这一点，在AID-/-鼠B细胞中引入一种AID-APOBEC 3G杂合体，该杂合体对在一系列C中的最后一个胞嘧啶脱氨基具有强烈的偏好，并将研究所产生的超突变、同种型转换和染色体易位.已知c-myc基因易位至IG基因之一需要AID，并且将鉴定和测序由突变体AID构建体促进的这种易位的连接。AID也将被突变以改变其将DNA中的5-甲基胞嘧啶（mC）转化为胸腺嘧啶的低效率。这种转化与DNA去甲基化有关，DNA去甲基化对胚胎细胞的重编程至关重要，并且与导致许多人类淋巴恶性肿瘤的易位热点有关。突变体的去甲基化活性将被证实，并且具有改变的mC偏好的AID突变体将在AID-/- B细胞中表达，以确定染色体易位的频率、谱或位置是否被改变。这些研究将创造新的工具来研究AID促进的遗传不稳定性，并将回答有关AID在核苷酸水平上如何选择DNA底物影响其有益和有害生物效应的具体问题。

DESCRIPTION (provided by applicant): Activation-induced deaminase (AID) belongs to the APOBEC family enzymes which convert cytosines in nucleic acids to uracil. AID is required for two processes essential for antibody maturation- somatic hypermutations and class-switch recombination Additionally, AID is also known to cause hypermutations in oncogenes and promote chromosome translocations that are hallmark of cancer. Among the unanswered questions regarding the role of AID in carcinogenesis is how AID selects certain genes and some loci for deamination. During the last funding period we studied the role played by transcription in the targeting process; in this proposal we will investigate how substrate selection by this enzyme affects the genetic outcomes. We will determine how the frequency, location and spectrum of genetic alterations changes when the substrate specificity of AID is altered. To accomplish this an AID- APOBEC3G hybrid with a strong preference for deaminating the last cytosine in a run of C's will be introduced in AID-/- murine B cells and the resulting hypermutations, isotype switching and chromosome translocations will be studied. The translocation of c-myc gene to one of the Ig genes is known to require AID and the junctions of such translocations promoted by mutant AID constructs will be identified and sequenced. AID will also be mutated to change its poor efficiency of converting 5-methylcytosine (mC) in DNA to thymine. Such conversions have been tied to DNA demethylation that is essential for reprogramming of embryonic cells and to translocation hotspots responsible for a number of human lymphoid malignancies. The demethylation activity of the mutants will be confirmed and AID mutants with altered preference for mC will be expressed in AID-/- B cells to determine whether the frequency, spectrum or locations of chromosome translocations are altered. These studies will create novel tools to study genetic instability promoted by AID and will answer specific questions regarding how the selection of DNA substrates by AID at nucleotide level influences its beneficial as well as harmful biological effects.