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

工作总结：当整合到人类基因组中时，HIV-1 基因组由RNA聚合酶II转录， 和病毒基因组RNA。 我们的目标是检查转录是否由 RNA聚合酶II可能有助于观察到的高变异， HIV基因组的某些部分。 为了解决这个问题，我们有 开发了一种策略来测量RNA转录的保真度， 聚合酶，包括纯化的T7 RNA聚合酶（RNAP）和RNA pol II， 人类细胞提取物。 T7 RNAP的结果表明， 应用的策略允许转录的定量评估 保真度，提供体外试验期间错误率的首次测量 在所有四种核苷酸存在下， template. 结果表明，转录的保真度是 对核苷酸库不平衡敏感，并提供直接证据， 局部序列上下文对错误产生有显著影响 在转录过程中。 我们还应用这一策略研究了损伤旁路的保真度 RNA聚合酶。 我们已经检查了T7 RNAP过去的转录， 特异性腺嘌呤残基与（+）抗反式或（-）抗反式加合 苯并[a]芘二醇环氧化物（BPDE）。 BPDE，一种常见的 环境污染物苯并[a]芘是一种公认的诱变剂， 致癌物质 为了测量旁路合成的保真度， 开发了回复突变测定法。 在赭石密码子T的对面， 转录链含有N6-BPDE加合或非加合的 腺嘌呤T7 RNAP在32%的时间内绕过（-）抗反式BPDE，而（+）抗- trans-BPDE 18%。 旁路合成是高度致突变的， （-）抗反式BPDE旁路的逆转频率升高 12，000倍，（+）抗反式-BPDE旁路6，000倍，相对于 未加合模板上转录的逆转频率。 腺嘌呤被错误插入优先相反的加合物，其次是 鸟嘌呤的错误插入

Summary of Work: When integrated in to the human genome, the HIV-1 genome is transcribed by the RNA polymerase II to produce both messenger and viral genomic RNAs. Our goal is to examine whether transcription by RNA polymerase II may contribute to the high variation observed in certain portions of the HIV genome. To adress this question we have developed a strategy to measure the fidelity of transcription by RNA polymerases including purified T7 RNA polymerase (RNAP)and RNA pol II in human cell extracts. The results with T7 RNAP demonstrate that the applied strategy allowes a quantitative evaluation of transcription fidelity, providing the first measurements of error rates during in vitro transcription in the presence of all four nucleotides on a natural template. The results indicate that the fidelity of transcription is sensitive to a nucleotide pool imbalance and provide direct evidence that local sequence contexts have a pronounced effect on error production during transcription. We have also applied this strategy to study the fidelity of damage bypass by RNA polymerases. We have examined transcription by T7 RNAP past a specific adenine residue adducted with (+)anti-trans- or (-) anti-trans- benzo[a]pyrene diol epoxide (BPDE). BPDE, a metabolite of a common environmental pollutant, benzo[a]pyrene, is an established mutagen and carcinogen. To measure the fidelity of bypass synthesis, an ochre codon reversion assay was developed. Opposite the T of the ochre codon, the transcribed strand contained the N6-BPDE adducted- or a non-adducted adenine. T7 RNAP bypassed (-)anti-trans-BPDE 32% of the time and (+)anti- trans-BPDE 18% of the time. The bypass synthesis was highly mutagenic, with reversion frequency for bypass of (-)anti-trans-BPDE elevated 12,000-fold and that for (+)anti-trans-BPDE bypass 6,000-fold relative to the reversion frequency of transcription on the unadducted template. Adenine was misinserted preferentially opposite the adduct, followed by misinsertion of guanine.