1887

Abstract

Moloney murine leukemia virus (M-MuLV) integrase (IN) catalyses the insertion of the viral genome into the host chromosomal DNA. The limited solubility of the recombinant protein produced in led the authors to explore the use of for expression of M-MuLV IN. IN was expressed in yeast and purified by chromatography on nickel–NTA agarose. IN migrated as a single band in SDS-PAGE and did not contain IN degradation products. The enzyme was about twofold more active than the enzyme purified from and was free of nucleases. Using the yeast system, the substitution of the putative catalytic amino acid Asp184 by alanine was also analysed. The mutated enzyme was inactive in the assays. This is the first direct demonstration that mutation of Asp184 inactivates M-MuLV IN. Finally, was used as a model to assess the ability of M-MuLV IN to interact with eukaryotic protein partners. The expression of an active M-MuLV IN in yeast strains deficient in RAD52 induced a lethal effect. This phenotype could be attributed to cellular damage, as suggested by the viability of cells expressing inactive D184A IN. Furthermore, when active IN was expressed in a yeast strain lacking the ySNF5 transcription factor, the lethal effect was abolished, suggesting the involvement of ySNF5 in the cellular damage induced by IN. These results indicate that could be a useful model to study the interaction of IN with cellular components in order to identify potential counterparts of the natural host.

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2005-09-01
2019-11-14
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