Biochemical Basis of Somatic Hypermutation

描述（由申请人提供）：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免受各种各样的传染性病原体的侵害。这项资助的主要目的是研究人类免疫多样性的生化基础。从较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）和类别转换重组（CSR）。两种抗体多样化事件都需要最近发现的酶活化诱导的胞苷脱氨酶（AID）的作用。在过去的一年里，研究表明AID催化单链DNA底物上的胞嘧啶脱氨为尿嘧啶。在体外观察到的脱氨基最常发生在对应于体内SHM热点序列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，是了解SHM的生化基础。我们建议首先通过表征纯化的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脱氨基特异性的AID突变体，其不再有利于热点基序。我们将测试野生型AID特性是由其扫描DNA前体的能力决定的这一假设，与表现出降低的脱氨特异性的进行性较低的AID突变体相反。只有活跃转录的免疫球蛋白可变基因经历SHM。在第三个具体的目标，我们将研究艾滋病催化脱氨基使用真核转录系统，包括RNA聚合酶II和转录因子。使用“经典”的生化加回实验，我们将进行一个系统的搜索转录组件的功能，目标艾滋病的转录装置。前三个具体的目的是为了澄清AID在启动C到T的转换突变的非转录的立场，在一个重要的类的热点基序在SHM的作用。具体目标4中体现的一个长期目标是研究突变链特异性，包括A和T序列的突变。这个目标将调查艾滋病与碱基切除修复，错配修复和最近发现的易错DNA聚合酶。

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.