scholarly journals Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it.

1989 ◽  
Vol 9 (12) ◽  
pp. 5373-5386 ◽  
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Base Pair ◽  
Regulatory Region ◽  
Small Subunit ◽  
Heterologous Promoter ◽  
Cellular Factors ◽  
Dna Binding Factors ◽  
Responsive Element

The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis. Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication. Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region. A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter. This fragment contained both positively and negatively acting sequences. Four DNA-binding factors interacted with the RNR2 regulatory region. One factor was identified as the GRF1 protein, the product of the RAP1 gene. GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment. UAS2 activity was repressed by the 42-bp fragment. Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter. These factors are likely to mediate the response of RNR2 to DNA damage.

Identification of the DNA damage-responsive element of RNR2 and evidence that four distinct cellular factors bind it

1989 ◽  
Vol 9 (12) ◽  
pp. 5373-5386
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Base Pair ◽  
Regulatory Region ◽  
Small Subunit ◽  
Heterologous Promoter ◽  
Cellular Factors ◽  
Dna Binding Factors ◽  
Responsive Element

The RNR2 gene encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of the deoxyribonucleotides needed for DNA synthesis. Transcription of this gene is induced approximately 20-fold in response to environmental stimuli that damage DNA or block DNA replication. Deletion and subcloning analysis identified two, and possibly three, upstream activating sequences (UAS) and one repressing (URS) element in the RNR2 regulatory region. A 42-base-pair (bp) fragment from this region was found to be necessary for proper regulation of RNR2 and to be capable of conferring DNA damage inducibility upon a heterologous promoter. This fragment contained both positively and negatively acting sequences. Four DNA-binding factors interacted with the RNR2 regulatory region. One factor was identified as the GRF1 protein, the product of the RAP1 gene. GRF1 bound to the UAS2 element of RNR2, which was found to be directly adjacent to the 42-bp fragment. UAS2 activity was repressed by the 42-bp fragment. Three other factors bound to the 42-bp fragment; one of these factors, RRF3, had a second binding site in the RNR2 promoter. These factors are likely to mediate the response of RNR2 to DNA damage.

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability

1987 ◽  
Vol 7 (8) ◽  
pp. 2783-2793
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Cross Reaction ◽  
Expression Library ◽  
Gene Encoding ◽  
Ribonucleotide Reductases ◽  
Colony Color

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate

1987 ◽  
Vol 7 (10) ◽  
pp. 3673-3677
Author(s):  
H K Hurd ◽  
C W Roberts ◽  
J W Roberts
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Alkylating Agent ◽  
Small Subunit ◽  
Methyl Methanesulfonate

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

scholarly journals Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability.

1987 ◽  
Vol 7 (8) ◽  
pp. 2783-2793 ◽  
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Cross Reaction ◽  
Expression Library ◽  
Gene Encoding ◽  
Ribonucleotide Reductases ◽  
Colony Color

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

scholarly journals Identification of the gene for the yeast ribonucleotide reductase small subunit and its inducibility by methyl methanesulfonate.

1987 ◽  
Vol 7 (10) ◽  
pp. 3673-3677 ◽  
Author(s):  
H K Hurd ◽  
C W Roberts ◽  
J W Roberts
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Alkylating Agent ◽  
Small Subunit ◽  
Methyl Methanesulfonate

We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomyces cerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by DNA damage.

scholarly journals Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase

2017 ◽  
Vol 38 (6) ◽  
Author(s):  
Xiaorong Wu ◽  
Xiuxiang An ◽  
Caiguo Zhang ◽  
Mingxia Huang
Keyword(s):  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
S Phase ◽  
Nuclear Retention ◽  
Content Type ◽  
Dntp Pool ◽  
Checkpoint Kinases ◽  
Enhanced Sensitivity

ABSTRACTA tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. InSaccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin–cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion ofCLB6or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

scholarly journals Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 6105-6113 ◽  
Author(s):  
M Huang ◽  
S J Elledge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Essential Gene ◽  
Large Subunit ◽  
Signal Transduction Pathway ◽  
Transcriptional Response ◽  
Small Subunit ◽  
Cell Extracts

Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an alpha2beta2 tetramer consisting of two large and two small subunits. RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae. We have cloned a second essential gene encoding a homologous small subunit, RNR4. RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced. The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes. RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts. Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes. Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.

DNA damage induction of ribonucleotide reductase

1989 ◽  
Vol 9 (11) ◽  
pp. 4932-4940
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Ribonucleotide Reductase ◽  
Uv Light ◽  
Small Subunit ◽  
Lacz Fusion ◽  
Dna Damaging Agents ◽  
Yeast Strains ◽  
Increased Sensitivity ◽  
Beta Galactosidase

RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous.

scholarly journals DNA damage induction of ribonucleotide reductase.

1989 ◽  
Vol 9 (11) ◽  
pp. 4932-4940 ◽  
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Dna Damage ◽  
Stress Response ◽  
Ribonucleotide Reductase ◽  
Uv Light ◽  
Small Subunit ◽  
Lacz Fusion ◽  
Dna Damaging Agents ◽  
Yeast Strains ◽  
Increased Sensitivity ◽  
Beta Galactosidase

RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous.

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