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Journal of Bacteriology, February 2009, p. 1169-1179, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01145-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions ^,

Inna Grinberg,¹ Tatyana Shteinberg,¹ A. Quamrul Hassan,² Yair Aharonowitz,¹ Ilya Borovok,^1* and Gerald Cohen^1*

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel,¹ Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts²

Received 14 August 2008/ Accepted 24 November 2008

Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations.

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* Corresponding authors. Mailing address for G. Cohen: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. Phone: 972 3 6409649. Fax: 972 3 6422245. E-mail: coheng{at}post.tau.ac.il. Mailing address for I. Borovok: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. Phone: 972 3 6407505. Fax: 972 3 6422245. E-mail: IlyaBo{at}tauex.tau.ac.il

Published ahead of print on 1 December 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, February 2009, p. 1169-1179, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01145-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.