Biochemical and Biophysical Research Communications
Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage
Section snippets
Materials and methods
Yeast cells, growth and EMS treatment. Cells were grown in YPD media. Wild-type strain MY377 (MAT-a ade2 ura3 leu2 trp1 his3 bar1) and rad53-11 mutant strain MY376 (MAT-a ADE2 ura3 leu2 trp1 his3 rad53-11 pep4:URA3 bar1) have been previously described [7].
Ethylmethylsulfonate (EMS) mutagenesis was done as described by Machado et al. [8].
dNTP pool measurements. Approximately 3 × 108 cells were harvested and extracted as described by Muller [9]. Each precipitated sample was resuspended in 200 μl of
Results
To assess the role of the Rad53 pathway in dNTP accumulation, we first tested whether EMS treatment would elevate the size of the dNTP pools in wild-type cells. Asynchronously growing wild-type cells (MY377) were treated with 0.01% EMS, a DNA alkylating agent, and levels of all four dNTPs were measured. As shown in Fig. 1A, the dNTP pools in untreated cells were 256 ± 8 pm/108 cells for dTTP, 476 ± 44 pm/108 cells for dATP, 153 ± 8 pm/108 cells for dCTP and 130 ± 8 pm/108 cells for dGTP. After 1-h EMS
Discussion
In yeast, the convergence of many different RNR regulatory mechanisms to the Rad53 pathway highlights the role of the pathway in dNTP synthesis in response to DNA damage. However, there has been no study showing a direct relationship between the level of dNTPs and the Rad53 pathway. Our study revealed that the Rad53 pathway is necessary for dNTP accumulation in response to DNA damage.
It has been shown that cell survival after DNA damage is directly linked to elevated levels of dNTPs in yeast
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