Biochemical Basis of Somatic Hypermutation

描述（由申请人提供）：人类健康方面最重要的科学挑战之一是了解免疫系统如何能够抵御各种感染因子。这项拨款的主要目的是研究人类免疫多样性的生化基础。从低特异性抗体产生高亲和力抗体需要两个关键事件：体细胞超突变（SHM）和类开关重组（CSR）。这两种抗体多样化事件都需要最近发现的一种酶的作用，即激活诱导胞苷脱氨酶（AID）。在过去的一年中，已经证明AID在单链DNA底物上催化胞嘧啶脱氨为尿嘧啶。体外观察到的脱胺最常发生在体内与SHM热点序列对应的特定三核苷酸序列上。我们的具体目标，包含在四个具体目标中，是了解SHM的生化基础。我们建议首先通过表征纯化AID在热点和冷点序列中的脱氨特异性来解决这一问题。我们将进行动力学分析以确定人类野生型AID的结合和催化偏好。然后，我们将研究具有改变的C脱胺特异性的AID突变体，这些突变体不再支持热点基序。我们将验证野生型AID的特性是由其扫描DNA的能力决定的这一假设，而与此相反，较弱的AID突变体表现出较低的脱胺特异性。只有活跃转录的免疫球蛋白可变基因才会发生SHM。在第三个具体目标中，我们将利用真核转录系统，包括RNA聚合酶II和转录因子，研究艾滋病催化的脱胺反应。使用“经典的”生化加回实验，我们将进行系统的搜索转录成分，其功能是靶向AID到转录装置。前三个具体目标旨在阐明AID在非转录林分（SHM中一类重要的热点基序）中启动C到T转变突变的作用。具体目标四的长期目标是研究突变链特异性，包括A和T序列的突变。本研究将研究AID与碱基切除修复、错配修复和最近发现的易出错DNA聚合酶的结合。

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.