Activation-induced Deaminase and Antibody Maturation

描述（由申请人提供）：激活诱导脱氨酶（AID）属于APOBEC家族酶，可将核酸中的胞嘧啶转化为尿嘧啶。在抗体成熟的两个重要过程——体细胞超突变和类开关重组中，AID是必需的。此外，已知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。在关于AID在癌变中的作用的未解决的问题中，AID是如何选择某些基因和一些位点进行降解的。在上一次融资期间，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aids的底物特异性改变时，基因改变的频率、位置和频谱是如何变化的。为了实现这一目标，我们将在AID-/-小鼠B细胞中引入AID- APOBEC3G杂交体，该杂交体具有强烈的对C序列中最后一个胞嘧啶脱胺的偏好，并将研究AID-/-小鼠B细胞中产生的超突变、同型转换和染色体易位。已知c-myc基因向其中一个Ig基因的易位需要AID，并且将鉴定和测序由AID突变构建体促进的这种易位的连接。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，并与许多人类淋巴细胞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，对mC偏好改变的AID突变体将在AID-/- B细胞中表达，以确定染色体易位的频率、频谱或位置是否发生改变。这些研究将为研究AID促进的遗传不稳定性创造新的工具，并将回答有关AID在核苷酸水平上对DNA底物的选择如何影响其有益和有害的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.