Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans

Abstract

A balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts. IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

Keywords: Cryptococcus neoformans; DNA damage stress; DNA replication stress; ribonucleotide reductase.

FIG 1

Rnr1 and Rnr21, not Rnr22, are required for viability in C. neoformans. (A) Fold change of RNR1, RNR21, and RNR22 in P_CTR4:RNR1, P_CTR4:RNR21, and P_CTR4:RNR22 strains in the presence of BCS or Cu²⁺. Statistical significance of differences was determined by one-way analysis of variance (ANOVA) with Bonferroni’s test. Error bars indicate standard error of the mean (***, P < 0.001). (B) RNR1 and RNR21 are required for viability. Strains (B: WT, P_CTR4:RNR1, P_CTR4:RNR21, and P_CTR4:RNR22 promoter replacement strains; D: WT, P_CTR4:RNR21, and P_CTR4:RNR21 P_H3:RNR22) were cultured in a liquid YPD medium. The strains were 10-fold serially diluted and spotted onto YNB medium or YPD medium containing the indicated concentration of CuSO₄ and BCS. Strains were further incubated at 30°C for 4 days and photographed. (C) The constitutive overexpression of RNR22 in the P_CTR4:RNR21 strain. Total RNA was isolated from WT, P_CTR4:RNR21, and P_CTR4:RNR21 P_H3:RNR22 strains (KW1458 and KW1459) and cDNA was synthesized from these total RNA samples. Statistical significance of differences was determined by one-way analysis of variance (ANOVA) with Bonferroni’s test. Error bars indicate standard deviation (*** P < 0.001). (D) Complementation of reduced viability through RNR22 overexpression.

FIG 2

Expression levels of RNR1, RNR21, and RNR22 after HU treatment. (A) Expression of RNR1 and RNR21 was regulated by both Rad53 and Chk1. (B) Ssn6-Tup1 complex suppressed RNR22 expression. Quantitative RT-PCR analysis was performed using cDNA synthesized from the total RNA isolated from WT H99, rad53Δ, chk1Δ, ssn6Δ, tup1Δ, and rad53Δ chk1Δ double mutant treated with 50 mM HU. Three independent biological samples were analyzed with technical duplicates. Error bars indicate standard error of the mean (S. E. M). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant). (C) The ssn6Δ and tup1Δ mutants were sensitive to HU. The strains were cultured in liquid yeast extract peptone dextrose (YPD) medium, which was serially diluted and spotted onto the YPD medium containing HU (50 mM). The strains were further incubated at 30°C and photographed daily.

FIG 3

Bdr1 and Mbs1 cooperatively regulated expression levels of RNR1 and RNR21 genes under HU treatment. (A and B) Expression of MBS1, RNR1, RNR21, and RNR22 in the signaling mutants under HU treatment. qRT-PCR analysis was performed using cDNA synthesized from total RNA isolated from WT H99, rad53Δ, chk1Δ, rad53Δ chk1Δ double mutant, bdr1Δ, mbs1Δ, and bdr1Δ mbs1Δ double mutant treated with HU 50 mM. Three independent biological samples were analyzed with duplicate technical replicates. Error bars indicate standard error of the mean (S. E. M). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant). (C) The deletion of both BDR1 and MBS1 resulted in synergistic growth defects in response to HU. Strains were cultured in a liquid YPD medium and were serially diluted and spotted onto the YPD medium containing the indicated concentration of HU. Strains were further incubated at 30°C and photographed daily. (D) Constitutive overexpression of RNR1 in mbs1Δ P_H3:RNR1 strain in the presence or absence of HU. (E) Overexpression of RNR1 in mbs1Δ mutant resulted in increased HU sensitivity.

FIG 4

The expression levels of RNR1, RNR21, and RNR22 genes in response to DNA damage stress. (A) Expression of RNR1, RNR21, and RNR22 in WT upon 4-NQO or MMS treatment or gamma radiation exposure. (B and C) Expression levels of RNR1, RNR21, and RNR22 in WT, rad53Δ, chk1Δ, rad53Δ chk1Δ double mutant, bdr1Δ, mbs1Δ, and bdr1Δ mbs1Δ double mutants under MMS treatment. The qRT-PCR analysis was performed using cDNA synthesized from total RNA isolated from WT H99, rad53Δ, chk1Δ rad53Δ chk1Δ double mutant, bdr1Δ, mbs1Δ, and bdr1Δ mbs1Δ double mutants treated with MMS 0.02%. Three independent biological samples were analyzed with duplicate technical replicates. Error bars indicate standard error of the mean (S. E. M). (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant).

FIG 5

Transcriptional changes in RNR21 resulted in increased sensitivity in response to DNA replication stress. (A) Expression of RNR1 and RNR21 in P_H3:RNR1 and P_H3:RNR21 strains in response to HU. qRT-PCR analysis was performed using cDNA synthesized from total RNA isolated from H99, P_H3:RNR1, and P_H3:RNR21 strains treated with 50 mM HU. (B) The P_H3:RNR21 strains were highly susceptible to HU. Strains were cultured in liquid YPD medium at 30°C for 16 h. The serially diluted cells were spotted onto the solid media containing the indicated concentration of HU. (C) The WT, P_CTR4:RNR1, and P_CTR4:RNR21 strains were cultured in liquid YPD medium at 30°C for 16 h. Next, the strains were 10-fold serially diluted and spotted on the YPD medium containing the indicated concentration of HU in the presence or absence of BCS. The cells were further incubated at 30°C for 3 days. Statistical significance of differences was determined by analysis of variance (ANOVA) with Bonferroni’s test (Prizm). Error bars indicate standard error of the mean (***, P < 0.001 and **, P < 0.01).

FIG 6

Proposed model of Rad53- and Chk1-dependent DNA replication and damage stresses. In response to HU treatment (DNA replication stress), Chk1, rather than Rad53, regulates RNR1 expression through the Mbs1 transcription factor. In contrast, Chk1 and Rad53 cooperatively control expression levels of RNR21 through Mbs1 and Bdr1 transcription factors. The Ssn6-Tup1 complex suppresses RNR22 expression. In response to MMS treatment (DNA damage stress), Chk1 and Rad53 equally contribute to RNR1 induction, whereas Rad53 and Chk1 play major and minor roles, respectively, in RNR21 induction. Chk1 and Rad53 are not involved in the regulation of RNR22 under both DNA replication and damage stress. Mbs1 plays a major role in MMS-mediated induction of RNR1, RNR21, and RNR22, whereas Bdr1 is involved in RNR21 and RNR22 induction in an opposite manner.