Abstract
Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.
Highlights
Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the rate-limiting step in the conversion of ribonucleotides to concomitant deoxyribonucleotides during de novo DNA synthesis [1, 2]
Ribonucleotide reductases (RNR) is a heterodimeric tetramer consisting of α2β2 heterodimers, where α2 denote the larger R1 subunit which serves as the catalytic site for rNTP reduction and β2 denote the R2 subunit which serves a regulatory role in conjunction with the R1 catalytic counterpart and function in S-phase DNA replication
The mechanism of RNR mediated catalysis is largely aided by redox modification S-glutathionylation which depends upon the availability of GSH-Grx electron donor systems
Summary
Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the rate-limiting step in the conversion of ribonucleotides (rNTP) to concomitant deoxyribonucleotides (dNTP) during de novo DNA synthesis [1, 2]. GSH formed adduct with the R1 subunit in a concentration-dependent manner presumably at a higher concentration (5–10 mM) with R1-R2 or R1p53R2 complex during the catalysis and provided electrons to the shuttle dithiols for efficient reduction of active site thiols, which would otherwise not be reduced by redoxins themselves due to sterically narrow active site pocket of R1 [8, 4] This novel mechanism allowed RNR to maintain its active site thiols in reduced state. S-glutathionylation has been reported to contribute to cancer progression and treatment resistance [10] This can provide a reasonable explanation why the GSH-Grx system is a more efficient electron donor for RNR disulfide reduction and sustain high RNR activity. We report Sglutathionylation of p53R2 subunit similar to R2 subunit
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