Activation-induced Deaminase and Antibody Maturation

描述(申请人提供)：激活诱导脱氨酶(AID)属于APOBEC家族酶，能将核酸中的胞嘧啶转化为尿嘧啶。抗体成熟所必需的两个过程--体细胞超突变和类别切换重组--都需要辅助。此外，众所周知，AID还会导致癌基因的超突变，并促进作为癌症标志的染色体易位。在有关AID在癌症发生中的作用的悬而未决的问题中，有一个问题是AID如何选择某些基因和某些位点进行脱氨。在上一次资助期间，我们研究了转录在靶向过程中所起的作用；在这项提议中，我们将研究这种酶对底物的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，基因改变的频率、位置和频谱如何改变。为了实现这一点，将在AID-/-小鼠B细胞中引入AID-APOBEC3G杂交种，该杂交种强烈倾向于脱氨C‘s中的最后一种胞嘧啶，并将研究由此产生的超突变、同型转换和染色体易位。已知c-myc基因到免疫球蛋白基因之一的易位需要AID，突变的AID结构将促进这种易位的连接并进行鉴定和测序。AID也将发生突变，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。这种转化与DNA去甲基化有关，DNA去甲基化对于胚胎细胞的重新编程和导致许多人类淋巴系统恶性肿瘤的易位热点是必不可少的。将证实突变体的去甲基化活性，并在AID-/-B细胞中表达对MC具有改变偏好的AID突变体，以确定染色体易位的频率、谱或位置是否改变。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在核苷酸水平上选择DNA底物如何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.