Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase
Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase
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DOI：
10.1096/fj.08-115352
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发表时间：
2009-01-01
期刊：
影响因子：
4.8
通讯作者：
Bhagwat, Ashok S.
中科院分区：
文献类型：
--
作者：
Canugovi, Chandrika;Samaranayake, Mala;Bhagwat, Ashok S.
重排免疫球蛋白基因的转录和酶激活诱导脱氨酶(AID)的表达是抗体成熟过程中该基因体细胞超突变所必需的。而AID则是一种单链DNA-胞嘧啶脱氨酶，产生U。G错配会导致突变，但转录在这一过程中所起的作用还不太清楚。我们利用T7RNA聚合酶(RNAP)在AID存在下对Kan基因的体外转录和卡那霉素抗性的基因逆转试验来研究体细胞超突变过程中多簇突变(MCM)的原因。我们发现，根据转录条件的不同，AID可以引起单碱基替换或MCM。当使用野生型RNAP在生理浓度的核糖核苷三磷酸(NTPs)下进行转录时，几乎没有发现MCM。相反，通过降低NTP浓度或使用突变的RNAP来减缓延伸速度，会使含有MCM的回复株的百分比增加几倍。通过快速去除NTPs来阻止伸长复合体的形成，会导致RNA-DNA杂交物(R-环)的形成。用AID处理这些结构会导致使用MCMS产生高比例的Kan(R)反转剂。此外，选择在获得卡那霉素抗性期间发生突变的密码子附近停滞的转录延伸复合体会导致绝大多数MCM的回复突变。这些结果表明，如果RNAP II在免疫球蛋白基因转录过程中暂停或停顿，AID很可能促进MCMS。由于生理条件的改变，如某些DNA一级或二级结构或DNA加合物的出现，已知会导致哺乳动物细胞的转录暂停和停滞，这一过程可能会在体细胞过度突变期间导致MCMS。-Canugoi，C.，Samaranayake，M.，Bhagwat，A.S.转录停顿和停滞通过人类激活诱导的脱氨酶导致多个聚集性突变。FASE B J.23，34-44(2009)
Transcription of the rearranged immunoglobulin gene and expression of the enzyme activation-induced deaminase (AID) are essential for somatic hypermutations of this gene during antibody maturation. While AID acts as a single-strand DNA-cytosine deaminase creating U . G mispairs that lead to mutations, the role played by transcription in this process is less clear. We have used in vitro transcription of the kan gene by the T7 RNA polymerase (RNAP) in the presence of AID and a genetic reversion assay for kanamycin-resistance to investigate the causes of multiple clustered mutations (MCMs) during somatic hypermutations. We find that, depending on transcription conditions, AID can cause single-base substitutions or MCMs. When wild-type RNAP is used for transcription at physiologically relevant concentrations of ribonucleoside triphosphates (NTPs), few MCMs are found. In contrast, slowing the rate of elongation by reducing the NTP concentration or using a mutant RNAP increases several-fold the percent of revertants containing MCMs. Arresting the elongation complexes by a quick removal of NTPs leads to formation of RNA-DNA hybrids (R-loops). Treatment of these structures with AID results in a high percentage of Kan(R) revertants with MCMs. Furthermore, selecting for transcription elongation complexes stalled near the codon that suffers mutations during acquisition of kanamycin-resistance results in an overwhelming majority of revertants with MCMs. These results show that if RNAP II pauses or stalls during transcription of immunoglobulin gene, AID is likely to promote MCMs. As changes in physiological conditions such as occurrence of certain DNA primary or secondary structures or DNA adducts are known to cause transcriptional pausing and stalling in mammalian cells, this process may cause MCMs during somatic hypermutation.-Canugovi, C., Samaranayake, M., Bhagwat, A. S. Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase. FASEB J. 23, 34-44 (2009)