Mechanistic studies on ribonucleotide reductase regulation and DNA damage response-application in cancer research

Targeting and damaging the DNA have been extensively used as an anti-cancer therapy in clinics. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit of ribonucleotide reductase (RNR). This enzyme is one of the key enzyme that has been targeted for cancer treatment and is crucial for DNA replication, maintenance of a balanced dNTP pools and DNA repair. The fidelity of DNA replication requires an appropriate balance of dNTPs. DNA replication stress (RS) can be defined as incomplete single-stranded DNA, leading to either stalling, collapse of the replication fork or its slow replication progression. Many factors such as decrease in dNTP pools after treatment with ribonucletide reductase inhi-bitors such as hydroxyurea and gemcitabine contribute to RS.The aim of the project was to investigate if there is any correlation between allosteric regulation of this enzyme and genomic stability (dNTP pool imbalance). In Our study, by using site-directed mutagenesis technique, we created a mutation in loop2 in the large subunit of RNR (Y285A) and after purification of the protein, the allosteric regulation of the Saccharomyces cerevisiae RNR was studied.The results showed that the RNR Y285A activity has been reduced 3.07-fold and 12.3-fold in the presence of GDP and CDP, respectively in comparison with the wildtype RNR activity.The consequence of Y285A mutation and dNTP imbalanced on DNA replication will be discussed.In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as ATM and ATR inhibitors. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells. This will be very important role in the cancer field and might be used as an anti-cancer therapy in clinics.

Keywords:

DNA , anti-cancer therapy , DNA replication stress , ribonucleotide reductase