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Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase.

ABSTRACT: DNA replication stress is an inefficient DNA synthesis process that leads replication forks to progress slowly or stall. Two main factors that cause replication stress are alterations in pools of deoxyribonucleotide (dNTP) precursors required for DNA synthesis and changes in the activity of proteins required for synthesis of dNTPs. Ribonucleotide reductase (RNR), containing regulatory hRRM1 and catalytic hRRM2 subunits, is the enzyme that catalyzes the conversion of ribonucleoside diphosphates (NDP) to deoxyribonucleoside diphosphates (dNDP) and thereby provides dNTP precursors needed for the synthesis of DNA. Here, we demonstrate that either endogenous or exogenous expression of Bcl2 results in decreases in RNR activity and intracellular dNTP, retardation of DNA replication fork progression, and increased rate of fork asymmetry leading to DNA replication stress. Bcl2 colocalizes with hRRM1 and hRRM2 in the cytoplasm and directly interacts via its BH4 domain with hRRM2 but not hRRM1. Removal of the BH4 domain of Bcl2 abrogates its inhibitory effects on RNR activity, dNTP pool level, and DNA replication. Intriguingly, Bcl2 directly inhibits RNR activity by disrupting the functional hRRM1/hRRM2 complex via its BH4 domain. Our findings argue that Bcl2 reduces intracellular dNTPs by inhibiting ribonucleotide reductase activity, thereby providing insight into how Bcl2 triggers DNA replication stress.

SUBMITTER: Xie M

PROVIDER: S-EPMC3887143 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase.

Xie Maohua M Yen Yun Y Owonikoko Taofeek K TK Ramalingam Suresh S SS Khuri Fadlo R FR Curran Walter J WJ Doetsch Paul W PW Deng Xingming X

Cancer research 20131106 1

DNA replication stress is an inefficient DNA synthesis process that leads replication forks to progress slowly or stall. Two main factors that cause replication stress are alterations in pools of deoxyribonucleotide (dNTP) precursors required for DNA synthesis and changes in the activity of proteins required for synthesis of dNTPs. Ribonucleotide reductase (RNR), containing regulatory hRRM1 and catalytic hRRM2 subunits, is the enzyme that catalyzes the conversion of ribonucleoside diphosphates ( ...[more]

PMID: 24197132

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Project description:Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an H2O2 addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (nrdJ). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. IMPORTANCE The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an H2O2 addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.

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Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase.

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Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase.

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