Studies of Ribonucleotide Reductase in Crucian Carp—An Oxygen Dependent Enzyme in an Anoxia Tolerant Vertebrate

Guro K Sandvik,Åsmund K Røhr,Jonas Bergan,K Kristoffer Andersson,Göran E Nilsson,Ane B Tomter,Giorgio Zoppellaro,Stian Ellefsen,Matthias Kolberg,Anne-Laure Barra,Anna Kristina Croft

doi:10.1371/journal.pone.0042784

Abstract

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O2) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.

Highlights

Crucian carp (Carassius carassius) has the extraordinary ability to live in the complete absence of oxygen for up to several months under the ice in small ponds [1,2]
Full-length cloning of crucian carp Ribonucleotide reductase (RNR) subunits Two paralogs of each RNR
The two RNR R1 paralogs, R1i (JQ679004) and R1ii (JQ679005) had coding domain sequences (CDS) that translated into products of 793 amino acid residues

Summary

Introduction

Crucian carp (Carassius carassius) has the extraordinary ability to live in the complete absence of oxygen (anoxia) for up to several months under the ice in small ponds [1,2] In part, this is made possible by its ability to produce ethanol as the end-product in glycolysis, avoiding lactate accumulation and acidosis [3]. Class II RNRs use deoxyadenosylcobalmin as cofactor instead of the R2 subunit and does not depend on oxygen for the generation of radical. This variant is found in different bacteria, and a few unicellular eukaryotes. With the help of an ironsulphur protein and S-adenosyl methionine the class III RNR found in some bacteria forms a glycyl radical that is lost in the presence of oxygen [9]

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Conclusion