Rapid cell division contributes to efficient induction of A/T mutations during Ig gene hypermutation
Rapid cell division contributes to efficient induction of A/T mutations during Ig gene hypermutation
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DOI：
10.1016/j.molimm.2011.06.218
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发表时间：
2011-09-01
影响因子：
3.6
通讯作者：
Wang, Ji-Yang
中科院分区：
文献类型：
--
作者：
Kano, Chie;Ouchida, Rika;Wang, Ji-Yang
IG基因超突变由激活诱导的胞苷脱氨酶（AID）启动，其将胞嘧啶转化为尿嘧啶并产生U：G损伤。未解之谜之一是艾滋病引发的U：G病变如何导致有效诱导生发中心（GC）B细胞V（H）基因中未受损A/T碱基的突变。遗传和生物化学证据表明，错配修复途径的组分和低保真度DNA聚合酶eta是诱导A/T突变所必需的。然而，错配修复熟练的NIH 3 T3细胞不能产生高频率的A/T突变，即使在DNA聚合酶eta过表达后，这表明涉及其他机制。由于GC B细胞在经历超突变的同时经历巨大的扩增，我们假设快速细胞分裂可能在A/T突变的诱导中起作用。为了验证这一假设，我们利用了一个有效的体外诱变系统，它密切反映了生理IG基因超突变，在人GC样B细胞系拉莫斯。将用AID-IRES-GFP逆转录病毒转导的拉莫斯细胞在补充有20%或2%胎牛血清（FBS）的培养基中培养10天，以分别允许快速和缓慢增殖。对V（H）基因突变的分析显示，与20% FBS相比，2% FBS中的A/T突变显著减少，转换比颠换受影响更大。这些结果表明，快速细胞分裂有助于有效诱导A/T突变，并表明DNA复制的速率对艾滋病触发的U：G病变的处理有深远的影响。(C)2011爱思唯尔有限公司保留所有权利。
Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase eta are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase eta overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions. (C) 2011 Elsevier Ltd. All rights reserved.