Wilson P C, de Bouteiller O, Liu Y J, Potter K, Banchereau J, Capra J D, Pascual V
Molecular Immunology Center, Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Texas 75235-9140, USA.
J Exp Med. 1998 Jan 5;187(1):59-70. doi: 10.1084/jem.187.1.59.
During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (approximately 110,000 nucleotides) from CD38+IgD- germinal center, and CD38-IgD- memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.
在生发中心反应过程中，免疫球蛋白V基因会引入随机突变，以增加抗体分子的亲和力，并进一步使B细胞库多样化。对抗原导向的、产生高亲和力表面免疫球蛋白的B细胞克隆进行选择，会导致抗体反应的亲和力成熟。Ig基因的突变通常是碱基对替换，不过据报道DNA插入和缺失也会以低频率发生。在本研究中，我们描述了在其他方面发生体细胞突变的VH基因cDNA分子中的5个插入事件和4个缺失事件。其中2个插入事件和所有4个缺失事件是通过对来自单个人类扁桃体的CD38+IgD-生发中心以及CD38-IgD-记忆B细胞群体的395个cDNA克隆（约110,000个核苷酸）进行测序获得的。在对扁桃体供体的基因组VH4库进行广泛表征后，未发现能够编码这6个cDNA克隆的种系基因。从其他来源分离出的这6个插入或缺失事件以及另外3个插入事件以三联体或其倍数形式出现，使转录本保持读框。此外，这些事件中的9个事件中有8个发生在互补决定区1（CDR1）或互补决定区2（CDR2），遵循与选择一致的模式，这使得这些事件不太可能是实验系统的假象。在未突变的IgD+CD38-滤泡套膜cDNA克隆中缺乏类似情况，从统计学上表明这些事件与体细胞超突变过程相关（P = 0.014）。对本文报道的9个插入/缺失事件以及从文献中收集的另外25个插入或缺失事件进行仔细审查表明，在大多数情况下，能够产生环状中间体的DNA序列中的二级结构元件可能是一个先决条件。此外，这些事件最常涉及类似于已知体细胞超突变内在热点的序列基序。这些插入/缺失事件与涉及缺乏校对能力的不稳定聚合酶复合物的体细胞超突变模型一致，并表明在超突变过程中V基因座处的DNA修复下调或改变。
