Atwood A, Choi J, Levin H L
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 1998 Feb;72(2):1324-33. doi: 10.1128/JVI.72.2.1324-1333.1998.
Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells. Schizosaccharomyces pombe is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts.
逆转录病毒及其亲属，即长末端重复序列逆转座子，拥有一种整合酶蛋白（IN），该蛋白是将逆转录产物插入宿主细胞基因组所必需的。粟酒裂殖酵母是Tf1的宿主，Tf1是一种具有整合活性的长末端重复序列逆转座子，可通过酵母遗传学技术进行研究。在本研究中，我们试图鉴定Tf1中特异性影响转座整合步骤的氨基酸替换。除了寻找IN中的氨基酸替换外，我们还探讨了其他Tf1蛋白对整合有贡献的可能性。通过比较监测转座和逆转录的遗传分析结果，我们能够在整个Tf1中寻找阻止转座但不阻止逆转录产物合成的点突变。这些Tf1的突变版本是在转座整合步骤中存在缺陷的元件候选物。发现Tf1中导致低水平整合的五个突变位于IN蛋白中：N端锌结构域中有两个替换，催化核心中有两个，C端结构域中有一个。这些结果表明，Tf1转座需要三个IN结构域中的每一个。讨论了这五个氨基酸残基在IN功能中的潜在作用。两个降低整合的突变映射到Tf1逆转录酶的核糖核酸酶H（RH）结构域。通过重组和DNA印迹分析确定，具有RH突变的Tf1元件产生高水平的逆转录产物。这些结果表明，Tf1的RH具有对转座至关重要的功能，该功能独立于逆转录产物的积累。
