RNR3 (DIN1, YIL066C) encodes the minor, DNA-damage-inducible large (R1, alpha) subunit of class Ia ribonucleotide reductase, about 80% identical to the essential major large subunit Rnr1. The large subunit carries the catalytic cysteines that reduce ribonucleoside diphosphates (ADP, GDP, CDP, UDP) to the corresponding 2'-deoxyribonucleoside diphosphates and the allosteric specificity and activity (ATP/dATP) sites that balance dNTP pools; the radical needed for catalysis is supplied by the Rnr2-Rnr4 small-subunit heterodimer. RNR3 is barely expressed in unperturbed cells but is strongly induced by DNA damage and replication stress through the Mec1-Rad53-Dun1 checkpoint and Crt1 derepression. It is not essential, and purified Rnr3 alone has less than 1% of the activity of Rnr1, but it is strongly activated when it forms a heterodimer with Rnr1, and high-copy RNR3 suppresses rnr1 lethality. Rnr3, like Rnr1, is cytoplasmic, where the active holoenzyme assembles with the small subunit.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IDA PMID:11893751 Yeast DNA damage-inducible Rnr3 has a very low catalytic act... | ACCEPT | Summary: Domkin et al. purified recombinant Rnr3 and measured CDP reduction with the Rnr2/Rnr4 small subunit: Rnr3 alone has <1% of Rnr1 activity but is strongly stimulated in an Rnr1/Rnr3 complex. 'contributes_to' correctly reflects that activity needs the radical-bearing small subunit. Reason: Direct biochemical evidence for the yeast protein; core activity of the alternative large subunit. Supporting Evidence: PMID:11893751 The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. PMID:11893751 a strong synergism between Rnr3 and Rnr1 was observed |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro IPR013509 (RNR large subunit N-terminal) / RHEA:23252 / EC 1.17.4.1 mapping. Rnr3 retains the catalytic machinery of the large subunit and has measurable, if low, intrinsic activity, so the family-level transfer is correct; in vivo it acts within the alpha2-beta-beta' holoenzyme. Reason: Correct mapping; consistent with the IDA contributes_to annotation. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt Catalyzes the biosynthesis of deoxyribonucleotides from the PMID:11893751 The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways (PWY-7222-1, GDP reduction) GO-CAM conversion lists RNR1-4 as the complexes catalysing NDP reduction. Correct for Rnr3 as a constituent of the damage-induced Rnr1/Rnr3 holoenzyme. Reason: Agrees with the IDA evidence; contributes_to is the right qualifier. Supporting Evidence: PMID:11893751 a strong synergism between Rnr3 and Rnr1 was observed |
| GO:0005524 ATP binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from PTN000171388 (RNR large subunit node; E. coli NrdA, M. tuberculosis NrdE). ATP binds the N-terminal ATP-cone activity site and the specificity site of class Ia large subunits as an allosteric activator. Rnr3 retains these sites (UniProt By similarity) but this is regulatory ligand binding, not the catalytic function. Reason: Biochemically plausible allosteric-effector binding; non-core. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt and activation sites on the large subunit. The type of nucleotide bound |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro IPR013509 mapping to ATP binding; reflects the allosteric ATP-cone of the large subunit. Correct but describes effector binding, not the reaction. Reason: Non-core regulatory ligand binding. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt and activation sites on the large subunit. The type of nucleotide bound |
| GO:0005737 cytoplasm | IDA PMID:12732713 Subcellular localization of yeast ribonucleotide reductase r... | ACCEPT | Summary: Yao et al. immunofluorescence: Rnr3, detectable only after HU/MMS or in crt1 cells, is predominantly cytoplasmic, like Rnr1, whereas Rnr2/Rnr4 are nuclear until S phase or damage. Reason: Direct localisation evidence for Rnr3; the cytoplasm is where the holoenzyme acts. Supporting Evidence: PMID:12732713 During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm PMID:12732713 Interestingly, under each of these conditions, Rnr3 was predominantly present in the cytoplasm |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SubCell mapping (SL-0086 Cytoplasm), itself based on PMID:12732713. Correct. Reason: Redundant with the IDA but correct. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:12732713}. |
| GO:0005737 cytoplasm | NAS PMID:16537480 Structures of eukaryotic ribonucleotide reductase I define g... | ACCEPT | Summary: NAS from the Rnr1 crystal-structure paper (Xu et al. 2006), which describes the eukaryotic large subunit; cytoplasmic location for Rnr3 is independently established by IDA. Reason: Statement is consistent with direct evidence; low added value but correct. Supporting Evidence: PMID:12732713 During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm |
| GO:0005737 cytoplasm | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways RCA (PWY-7222-1) places the RNR reaction in cytoplasm (is_active_in). Consistent with cytoplasmic Rnr1/Rnr3 and checkpoint-driven export of Rnr2-Rnr4 to assemble the holoenzyme there. Reason: Correct compartment for the active enzyme. Supporting Evidence: PMID:12732713 During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm |
| GO:0005739 mitochondrion | HDA PMID:14576278 The proteome of Saccharomyces cerevisiae mitochondria. | KEEP AS NON CORE | Summary: Detected in the Sickmann et al. purified-mitochondria MS proteome. Rnr3 is predominantly cytoplasmic by immunofluorescence and there is no evidence that ribonucleotide reduction occurs inside yeast mitochondria; detection probably reflects co-purification or outer-surface association of this abundant cytoplasmic protein. Reason: High-throughput organellar MS hit; retained as non-core, not a site of function. Supporting Evidence: PMID:12732713 During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm |
| GO:0005739 mitochondrion | HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen... | KEEP AS NON CORE | Summary: Second mitochondrial-proteome MS detection (Reinders et al. PROMITO). Same caveat: no supporting evidence for an intramitochondrial RNR in yeast; Rnr3 is cytoplasmic. Reason: High-throughput organellar MS hit; non-core. Supporting Evidence: PMID:12732713 During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm |
| GO:0005971 ribonucleoside-diphosphate reductase complex | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN000171388 with experimental support from RNR1 (SGD:S000000872), human RRM1, E. coli NrdA. Rnr3 is an alternative large subunit of the yeast RNR complex. Reason: Correct; Rnr3 forms Rnr1/Rnr3 heterodimers that join the holoenzyme. Supporting Evidence: PMID:11893751 We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme file:yeast/RNR3/RNR3-uniprot.txt heterodimer of RNR1 and the damage-inducible RNR3. |
| GO:0005971 ribonucleoside-diphosphate reductase complex | TAS PMID:10716984 Yeast ribonucleotide reductase has a heterodimeric iron-radi... | ACCEPT | Summary: TAS from Chabes et al. 2000, whose introduction and discussion describe Rnr3 as the second large subunit and show Rnr3 is almost inactive alone but augments activity with Rnr1. Reason: Author statement consistent with later direct evidence (PMID:11893751). Supporting Evidence: PMID:10716984 Highly purified recombinant Rnr3p, with an amino acid sequence identity of 80% compared with the Rnr1p, is almost inactive when assayed together with the H 6 Rnr2p/Rnr4p heterodimer. PMID:12732713 In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). |
| GO:0009263 deoxyribonucleotide biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the RNR large-subunit node; deoxyribonucleotide biosynthesis is the process in which the large subunit performs the reduction step. Reason: Core process for an RNR catalytic subunit. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt Catalyzes the biosynthesis of deoxyribonucleotides from the |
| GO:0009263 deoxyribonucleotide biosynthetic process | IDA PMID:5459124 Ribonucleotide reductase from Saccharomyces cerevisiae. | ACCEPT | Summary: Vitols et al. 1970 characterised ribonucleotide reductase activity from yeast extracts, before the subunit genes were known (abstract unavailable in cache); in normal cells this activity is mostly Rnr1-based. The process is nevertheless correct for Rnr3, which has measurable reductase activity and contributes to dNTP synthesis after DNA damage. Reason: Deferring to curator; process is supported for Rnr3 by PMID:11893751 and PMID:2199320. Supporting Evidence: PMID:11893751 The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. PMID:2199320 A high-copy-number clone of RNR3 is able to suppress the lethality of rnr1 mutations. |
| GO:0009263 deoxyribonucleotide biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: ARBA/InterPro IPR013509 mapping; correct family-level process for an RNR large subunit. Reason: Correct. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt Catalyzes the biosynthesis of deoxyribonucleotides from the |
| GO:0106387 'de novo' GMP biosynthetic process | RCA GO_REF:0000123 | REMOVE | Summary: YeastPathways RCA from PWY-7222-1 ('guanosine deoxyribonucleotides de novo biosynthesis II'), whose GO-CAM conversion tags the GDP reduction step as part_of 'de novo' GMP biosynthesis. RNR reduces GDP to dGDP; it does not participate in IMP-to-GMP synthesis. Reason: Pathway-to-GO mapping error: wrong process (GMP rather than deoxyribonucleotide biosynthesis), already covered by GO:0009263. Supporting Evidence: file:yeast/RNR3/RNR3-uniprot.txt Catalyzes the biosynthesis of deoxyribonucleotides from the |
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Download this section (compressed HTML)Q: Does Rnr3 contribute measurably to dNTP pools in vivo after DNA damage, given that rnr3 nulls have no obvious damage phenotype and Rnr3 levels stay below one-tenth of Rnr1?
Q: Is the Rnr3 detected in mitochondrial proteomes a contaminant, or is a fraction of RNR associated with the mitochondrial outer membrane to supply mtDNA synthesis?
Experiment: Measure dNTP pools and replication fork progression after MMS in rnr1 hypomorph strains with and without RNR3, and in strains expressing catalytically dead Rnr3 (active-site cysteine mutants).
Hypothesis: Rnr1/Rnr3 heterodimers increase dNTP output when Rnr1 is limiting
Type: genetics / dNTP measurement
Minor isoform of large subunit of ribonucleotide-diphosphate reductase; the RNR complex catalyzes rate-limiting step in dNTP synthesis, regulated by DNA replication and DNA damage checkpoint pathways via localization of small subunits; RNR3 has a paralog, RNR1, that arose from the whole genome duplication
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