Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

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Abstract

Deoxyribonucleotide pools are maintained at levels that support efficient and yet accurate DNA replication and repair. Rad53 is part of a protein kinase regulatory cascade that, conceptually, promotes dNTP accumulation in four ways: (1) it activates the transcription of ribonucleotide reductase subunits by inhibiting the Crt1 repressor; (2) it plays a role in relocalization of ribonucleotide reductase subunits RNR2 and RNR4 from nucleus to cytoplasm; (3) it antagonizes the action of Sml1, a protein that binds and inhibits ribonucleotide reductase; and (4) it blocks cell-cycle progression in response to DNA damage, thus preventing dNTP consumption through replication forks. Although several lines of evidence support the above modes of Rad53 action, an effect of a rad53 mutation on dNTP levels has not been directly demonstrated. In fact, in a previous study, a rad53-11 mutation did not result in lower dNTP levels in asynchronous cells or in synchronized cells that entered the S-phase in the presence of the RNR inhibitor hydroxyurea. These anomalies prompted us to investigate whether the rad53-11 mutation affected dNTP levels in cells exposed to a DNA-damaging dose of ethylmethyl sulfonate (EMS). Although dNTP levels increased by 2- to 3-fold in EMS treated wild-type cells, rad53-11 cells showed no such change. Thus, the results indicate that Rad53 checkpoint function is not required for dNTP pool maintenance in normally growing cells, but is required for dNTP pool expansion in cells exposed to DNA-damaging agents.

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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