Rnr4p, a novel ribonucleotide reductase small-subunit protein

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Ribonucleotide reductases (RNRs) are crucial enzymes for DNA synthesis, catalyzing deoxyribonucleotide formation.
• Eukaryotic RNRs form alpha2beta2 tetramers, with beta subunits containing a critical iron-tyrosyl radical complex.
• In yeast (Saccharomyces cerevisiae), RNR1 and RNR3 encode alpha subunits, and RNR2 encodes a beta subunit.

Purpose of the Study:

• To identify and characterize a novel gene encoding a beta subunit of ribonucleotide reductase in yeast.
• To investigate the function and properties of the newly identified RNR4 gene product (Rnr4p).

Main Methods:

• Gene identification and sequencing in Saccharomyces cerevisiae.
• Phenotypic analysis of rnr4 mutants, including hydroxyurea sensitivity and cell cycle arrest.
• Biochemical assays to measure ribonucleotide reductase activity in mutant extracts.
• Analysis of Rnr4p sequence for conserved functional domains and mutation studies.

Main Results:

• A second beta-subunit gene, RNR4, was identified in yeast.
• rnr4 mutants exhibit phenotypes consistent with impaired ribonucleotide reductase activity.
• Rnr4p is required for in vitro ribonucleotide reductase activity, which can be restored by adding exogenous Rnr4p.
• RNR4 expression is induced by DNA-damaging agents.
• Rnr4p lacks key iron-binding motifs and its critical tyrosine residue is not essential for function, suggesting catalytic inactivity.

Conclusions:

• Rnr4p is a novel, catalytically inactive component of the yeast ribonucleotide reductase complex.
• Despite its inactivity, Rnr4p plays an essential role in the overall function of the ribonucleotide reductase enzyme.
• The discovery of RNR4 expands our understanding of the complex regulation and composition of ribonucleotide reductases.