کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1929657 | 1050470 | 2012 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein](/preview/png/1929657.png)
Two mutant Escherichia coli RecA proteins were prepared in which the ATP active site residue, Ser240, was replaced with asparagine and lysine (these amino acids are found in the corresponding positions in other bacterial RecA proteins). The S240N mutation had no discernible effect on the ATP-dependent activities of the RecA protein, indicating that serine and asparagine are functionally interchangeable at position 240. The S240K mutation, in contrast, essentially eliminated the ability of the RecA protein to utilize ATP as a nucleotide cofactor. The [S240K]RecA protein was able to catalyze the hydrolysis of dATP, however, suggesting that the absence of the 2′-hydroxyl group reduced an inhibitory interaction with the Lys240 side chain. Interestingly, the [S240K]RecA protein was able to promote an efficient LexA cleavage reaction but exhibited no strand exchange activity when dATP was provided as the nucleotide cofactor. This apparent separation of function may be attributable to the elevated S0.5 value for dATP for the [S240K]RecA protein (490 μM, compared to 20–30 μM for the wild type and [S240N]RecA proteins), and may reflect a differential dependence of the LexA co-protease and DNA strand exchange activities on the nucleotide cofactor-mediated stabilization of the functionally-active state of the RecA–ssDNA complex.
► A mutant RecA protein was prepared in which Ser240 was replaced with lysine.
► [S240K]RecA catalyzes dATP hydrolysis but has no ATP hydrolysis activity.
► [S240K]RecA promotes LexA cleavage but has no strand exchange activity with dATP.
► The co-protease and recombinase reactions differ in their NTP cofactor dependence.
► The S240K mutation alters the nucleotide cofactor specificity of the RecA protein.
Journal: Biochemical and Biophysical Research Communications - Volume 421, Issue 3, 11 May 2012, Pages 527–531