Advanced Radiation Technology Institute, Korea Atomic Energy Research Institutegrid.418964.6, Jeongeup, Republic of Korea.
Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
Microbiol Spectr. 2022 Aug 31;10(4):e0104422. doi: 10.1128/spectrum.01044-22. Epub 2022 Jun 23.
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 (large RNR subunit) and (one small RNR subunit) were required for cell viability, but not (another small RNR subunit). overexpression compensated for the lethality of suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled and expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of during DNA damage stress. Furthermore, the expression of , not but and , was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that expression was mainly regulated by Mbs1, whereas 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. 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 , , and 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.
脱氧核苷酸（dNTPs）在细胞内的浓度平衡对于 DNA 复制和修复过程至关重要。在模式酵母酿酒酵母中，Mec1-Rad53-Dun1 激酶级联主要在 DNA 复制和 DNA 损伤应激期间调节核糖核苷酸还原酶（RNR）基因的表达。然而，在担子菌真菌中，RNR 的调控机制在 DNA 复制和损伤应激期间仍然难以捉摸。在这里，我们观察到 C. neoformans 中的 （大亚基）和 （一个小亚基）对于细胞活力是必需的，但 （另一个小亚基）不是。过表达 可以补偿 的抑制致死性。与酿酒酵母中 RNR 的调控机制不同，Rad53 和 Chk1 激酶在 DNA 损伤和 DNA 复制应激下协同或分歧地控制 和 的表达。特别是，这项研究表明，Chk1 主要在 DNA 复制应激期间调节 的表达，而 Rad53 而不是 Chk1，在 DNA 损伤应激期间对 的表达起着重要作用。此外，在 DNA 复制应激期间，不是 ，而是 Ssn6-Tup1 复合物抑制了 的表达。值得注意的是，我们观察到 表达主要受 Mbs1 调控，而 表达在 DNA 复制和损伤应激期间作为 Rad53 和 Chk1 的下游因子，由 Mbs1 和 Bdr1 协同控制。总的来说，与其他酵母相比，RNR 在 C. neoformans 中的调控在 DNA 复制和 DNA 损伤应激方面具有进化保守和分歧的特征。在 DNA 复制或损伤应激期间，提供适当水平的脱氧核苷酸三磷酸（dNTPs）并激活 DNA 修复机制至关重要。核糖核苷酸还原酶（RNRs）由大亚基和小亚基组成，是合成 dNTP 的必需物质。RNR 受到干扰会导致 dNTP 细胞内浓度失衡，从而降低 DNA 修复保真度。尽管它们的作用很重要，但 RNR 的功能和调控尚未在担子菌真菌中阐明。在这项研究中，我们发现 C. neoformans 中编码 RNR 亚基的 、 和 基因在细胞活力中的作用。此外，它们的表达水平在 DNA 复制和损伤应激下通过 Rad53-Chk1 途径和 Ssn6-Tup1 复合物被分歧地调节。因此，本研究为担子菌真菌中 RNR 基因对 DNA 复制和损伤应激的调控机制提供了新的认识。
