Ribonucleotide reductases (RNRs) play indispensable roles in both DNA replication and repair. RNRs catalyze the reduction of ribonucleoside 5-di-or triphosphates (NDPs or NTPs) to deoxyribonucleoside 5-di- or triphosphates (dNDPs or dNTPs). The reduction from NDPs (or NTPs) to dNDPs (or dNTPs) is the only pathway for the de novo synthesis of the monomeric building blocks of DNA. Epstein–Barr virus (EBV) is a ubiquitous oncogenic herpesvirus that infects over 90% of the human population. Through sequence alignment, we have found that EBV RNR seems distinct from other reported class I RNR subclasses. Thus we investigated the nature of EBV RNR cofactor by in vitro reconstitution followed by activity assay and spectroscopic characterization. Initially we purified EBV RNR β subunit and reconstituted tyrosyl radical on β subunit by adding Fe2+ and ascorbate to the lysate. To improve the solubility of α subunit, MBP tag containing EBV RNR α subunit was purified by one-step Ni-NTA column and then MBP tag was removed by digestion with Factor Xa. Then holoenzyme activity assay was performed by combining α and β subunits adopting HPLC method.
RNR is a established anti-cancer and antiviral drug target. Till date many drugs targeting RNR have been developed, some of them are in clinical use and others are in clinical trial. Given the essentiality of the metal center for the activity of RNR, characterizing the EBV RNR metal cofactor and further study of its biosynthesis will provide the basis for developing antiviral drugs specifically against EBV and other viruses which contains similar architecture of metallocofactor assembly. |