Biochemical Basis of Somatic Hypermutation

描述(申请人提供)：与人类健康相关的最重要的科学挑战之一是了解免疫系统如何能够保护自己免受各种感染性病原体的侵袭。这笔赠款的广泛目标是研究人类免疫多样性的生物化学基础。从低特异性抗体中产生高亲和力抗体需要两个关键事件，即体细胞超突变(SHM)和类切换重组(CSR)。这两种抗体多样化事件都需要最近发现的一种酶-激活诱导胞苷脱氨酶(AID)的作用。在过去的一年里，研究表明，在单链DNA底物上，AID催化胞嘧啶脱氨为尿嘧啶。体外观察到的脱氨基最常出现在与体内SHM热点序列相对应的特定三核苷酸序列中。我们的具体目标包括四个具体目标，就是了解SHM的生化基础。我们建议首先通过鉴定纯化的AID在热点和冷点序列中的脱氨基特异性来解决这个问题。我们将进行动力学分析，以确定人野生型AID的结合和催化偏好。然后，我们将研究具有改变的C脱氨基特异性的AID突变体，这些突变体不再偏爱热点基序。我们将检验这一假设，即野生型AID的特性取决于其连续扫描DNA的能力，而不是进行性较低的AID突变体表现出降低的脱氨基特异性。只有活跃转录的免疫球蛋白可变基因才会经历SHM。在第三个特定目标中，我们将使用包括RNA聚合酶II和转录因子在内的真核转录系统来研究AID催化的脱氨基反应。利用“经典的”生化加回实验，我们将进行系统的转录成分的搜索，这些转录成分的功能是将AID靶向转录装置。前三个特定目标旨在阐明AID在非转录立场上启动C到T转换突变中的作用，该突变是SHM中一类重要的热点基序。一个较长期的目标，体现在具体目标四，是研究突变链特异性，包括A和T序列的突变。这一目标将结合碱基切除修复、错配修复和最近发现的容易出错的DNA聚合酶来研究AID。

DESCRIPTION (provided by applicant): One of the most important human health-related scientific challenges is to understand how the immune system is able to protect against a wide variety of infectious agents. The broad objective of this grant is to investigate the biochemical basis of human immunodiversity. Two key events are required to produce high affinity antibodies from lower specificity antibodies, somatic hypermutation (SHM) and class switch recombination (CSR). Both antibody diversification events require the action of a recently discovered enzyme, activation induced cytidine deaminase (AID). Within the past year it has been shown that AID catalyzes deamination of cytosine to uracil on single-stranded DNA substrates. The deaminations observed in vitro occur most frequently in specific trinucleotide sequences corresponding to SHM hot spot sequences in vivo. Our specific goal, encompassed in four specific aims, is to understand the biochemical basis of SHM. We propose to address this problem first by characterizing the deamination specificity of purified AID in hotspot and coldspot sequences. We will carry out a kinetic analysis to determine binding and catalytic preferences for human wild type AID. We will then investigate AID mutants with altered C deamination specificity that no longer favor hotspot motifs. We will test the hypothesis that wild type AID properties are determined by its ability to scan DNA processively, in contrast to less processive AID mutants exhibiting reduced deamination specificity. Only actively transcribed immunoglobulin variable genes undergo SHM. In the third specific aim, we will investigate AID-catalyzed deamination using a eukaryotic transcription system, including RNA polymerase II and transcription factors. Using "classical" biochemical add-back experiments, we will perform a systematic search for transcription components that function to target AID to the transcription apparatus. The first three specific aims are intended to clarify the role of AID in initiating C to T transition mutations in the non-transcribed stand, at an important class of hotspot motifs in SHM. A longer-term goal, embodied in specific aim four, is to investigate mutational strand specificity, including mutations at A and T sequences. This aim will investigate AID in conjunction with base excision repair, mismatch repair and recently discovered error-prone DNA polymerases.