Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and provide the monomeric precursors required for DNA replication and DNA repair. The class I RNRs are composed of two subunits: the ?n subunit binds the four NDP substrates and the allosteric effectors (NTPs and dATP) that govern substrate specificity and turnover rate. The ?? subunit houses the essential diferric-tyrosyl radical (Y) cofactor required to initiate the chemically difficult reduction process on ?n. The active RNR complex is ?n?2. Regulation of RNRs is largely responsible for controlling the relative ratios and amounts of the dNTP pools, which is critical to the fidelity of DNA replication and repair. Loss of this control can lead to cell death, genetic instability, and in humans, a predisposition to cancer. RNR's central role in nucleic acid metabolism has made them the successful target in the treatment of a number of malignancies. Regulation of RNR activity occurs at multiple levels: transcriptionally, by control of the subcellular localization of ?n and ?2, by the binding of allosteric effectors (ATP, dNTPs to ?n), by control of protein degradation, by control of the concentration of the Y generated by the di-iron metallo-cofactor, and by small protein inhibitors. This proposal focuses on the regulation of the class I RNRs from E. coli and S. cerevisiae. Two regulatory mechanisms will be examined using an integration of biochemical and genetic approaches. The first and second specific aims are to elucidate the biosynthetic and maintenance (repair) pathways of the essential diferric-Y cofactor of ?2 in E. coli and S. cerevisiae. Experiments are presented to identify the assembly factors required for iron and reducing equivalent delivery. The Y of the cofactor is the target of hydroxyurea used in the treatment of hematologic malignancies and of triapine in phase II clinical trials. Thus understanding whether the clusters can be repaired can have dramatic outcomes clinically. The third specific aim in S. cerevisiae is to understand mechanism of the small proteins: Sml1 and the newly discovered Spd1, in RNR inhibition. This understanding could identify a new therapeutic target. The long-range goal is to understand quantitatively how all of the regulatory mechanisms are integrated to control cellular dNTPs pools under different growth conditions.

Public Health Relevance

Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Their regulation is essential for controlling dNTP pools, critical for the fidelity of DNA replication and repair. Two regulatory mechanisms are examined in this proposal;understanding these mechanisms could lead to new therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081393-03
Application #
7802291
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2008-07-02
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
3
Fiscal Year
2010
Total Cost
$400,735
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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Wei, Yifeng; Li, Bin; Prakash, Divya et al. (2015) A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase. Biochemistry 54:7019-28
Huang, Mingxia; Parker, Mackenzie J; Stubbe, JoAnne (2014) Choosing the right metal: case studies of class I ribonucleotide reductases. J Biol Chem 289:28104-11
Rhodes, DeLacy V; Crump, Katie E; Makhlynets, Olga et al. (2014) Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis. J Biol Chem 289:6273-87
Wei, Yifeng; Funk, Michael A; Rosado, Leonardo A et al. (2014) The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant. Proc Natl Acad Sci U S A 111:E3756-65
Wei, Yifeng; Mathies, Guinevere; Yokoyama, Kenichi et al. (2014) A chemically competent thiosulfuranyl radical on the Escherichia coli class III ribonucleotide reductase. J Am Chem Soc 136:9001-13
Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V et al. (2014) Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights. J Biol Chem 289:6259-72
Parker, Mackenzie J; Zhu, Xuling; Stubbe, JoAnne (2014) Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions. Biochemistry 53:766-76
Zhang, Yan; Li, Haoran; Zhang, Caiguo et al. (2014) Conserved electron donor complex Dre2-Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis. Proc Natl Acad Sci U S A 111:E1695-704
Zhang, Yan; An, Xiuxiang; Stubbe, Joanne et al. (2013) Investigation of in vivo roles of the C-terminal tails of the small subunit (??') of Saccharomyces cerevisiae ribonucleotide reductase: contribution to cofactor formation and intersubunit association within the active holoenzyme. J Biol Chem 288:13951-9

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