DO TRANSCRIPTION ERRORS BY RNA POL II CONTRIBUTE TO VARIATION IN HIV

工作总结：这项工作的开始是为了研究RNA聚合酶II的转录是否可能有助于在HIV基因组的某些部分观察到的高变异。为了解决这个问题，我们已经开发了一种体外系统，该系统允许定量评价在所有四种核苷酸存在下在天然模板上合成期间人细胞提取物中RNA聚合酶（包括纯化的T7 RNA聚合酶（RNAP）和RNA pol II）的保真度。最近，我们使用TAA-ochre密码子逆转试验来检测T7 RNA聚合酶（T7 RNAP）通过在N6位置与（-）反式或（+）反式苯并[a]芘二醇环氧化物（BPDE）加合的腺嘌呤残基的转录保真度。T7 RNAP能够转录过任一BPDE异构体以产生全长径流转录物。发现旁路的程度对于（-）反式异构体为32%，对于（+）反式异构体为18%。转录过去的两个加合物是高度致突变的。相对于未加合模板上转录的逆转频率，（-）反反式异构体的旁路合成的逆转频率升高11，000倍，（+）反式异构体的逆转频率升高6000倍。腺嘌呤被错误插入优先，其次是鸟嘌呤，相反的腺嘌呤加合物与BPDE异构体。虽然碱基替换错误是迄今为止加合模板上最常见的突变，但也观察到3-和6-碱基缺失。这些结果表明，转录错误，特别是关于损害旁路，可能有助于细胞的突变负担。- 细胞提取物，DNA聚合酶，HIV-1，突变，逆转录酶，RNA，信使

Summary of Work: This work was initiated to examine whether transcription by RNA polymerase II may contribute to the high variation observed in certain portions of the HIV genome. To address this question we have developed an in vitro system that allows quantitative evaluation of the fidelity of RNA polymerases including purified T7 RNA polymerase(RNAP) and RNA pol II in human cell extracts during synthesis on a natural template in the presence of all four nucleotides. Recently we have employed this system using a TAA-ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase (T7 RNAP) past an adenine residue adducted at the N6 position with (-)anti-trans- or (+)anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full length run-off transcripts. The extent of bypass was found to be 32 % for the (-)anti-trans-isomer and 18 % for the (+)anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass-synthesis of the (-)anti-trans isomer was elevated 11,000-fold and that of the (+)anti-trans isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, 3- and 6-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell. - Cell Extracts, DNA Polymerases, HIV-1, Mutagenesis, Reverse Transcriptase, RNA, Messenger