Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

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Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

Journal of Virology ◽

10.1128/jvi.63.8.3389-3398.1989 ◽

1989 ◽

Vol 63 (8) ◽

pp. 3389-3398 ◽

Cited By ~ 57

Author(s):

T D Chung ◽

J P Wymer ◽

C C Smith ◽

M Kulka ◽

L Aurelian

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protein Kinase ◽

Ribonucleotide Reductase ◽

Herpes Simplex Virus Type ◽

Kinase Activity ◽

Large Subunit ◽

Protein Kinase Activity ◽

Simplex Virus

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Structure-function studies of the large subunit of ribonucleotide reductase from Escherichia coli

Biochemical Society Transactions ◽

10.1042/bst0160091 ◽

1988 ◽

Vol 16 (2) ◽

pp. 91-94 ◽

Cited By ~ 12

Author(s):

OLLE NILSSON ◽

TOMAS LUNDQVIST ◽

SOLVEIG HAHNE ◽

BRITT-MARIE SJÖBERG

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Ribonucleotide Reductase ◽

Large Subunit

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The Transmembrane Domain of the Large Subunit of HSV-2 Ribonucleotide Reductase (ICP10) Is Required for Protein Kinase Activity and Transformation-Related Signaling Pathways That Result in ras Activation

Virology ◽

10.1006/viro.1994.1223 ◽

1994 ◽

Vol 200 (2) ◽

pp. 598-612 ◽

Cited By ~ 28

Author(s):

C.C. Smith ◽

J.H. Luo ◽

J.C.R. Hunter ◽

J.V. Ordonez ◽

L. Aurelian

Keyword(s):

Protein Kinase ◽

Signaling Pathways ◽

Ribonucleotide Reductase ◽

Kinase Activity ◽

Transmembrane Domain ◽

Large Subunit ◽

Protein Kinase Activity ◽

Ras Activation

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Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0611095104 ◽

2007 ◽

Vol 104 (7) ◽

pp. 2217-2222 ◽

Cited By ~ 21

Author(s):

Z. Zhang ◽

K. Yang ◽

C.-C. Chen ◽

J. Feser ◽

M. Huang

Keyword(s):

Ribonucleotide Reductase ◽

Large Subunit ◽

C Terminus

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Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.23.11322 ◽

1993 ◽

Vol 90 (23) ◽

pp. 11322-11326 ◽

Cited By ~ 20

Author(s):

S Björklund ◽

K Hjortsberg ◽

E Johansson ◽

L Thelander

Keyword(s):

Ribonucleotide Reductase ◽

Genomic Library ◽

Protein Complexes ◽

Specific Binding ◽

Transcriptional Start Site ◽

Large Subunit ◽

Mobility Shift ◽

Direct Role ◽

Encoding Gene ◽

R1 Gene

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.

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Multiple Controls Regulate the Expression of mobE, an HNH Homing Endonuclease Gene Embedded within a Ribonucleotide Reductase Gene of Phage Aeh1

Journal of Bacteriology ◽

10.1128/jb.00321-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4648-4661 ◽

Cited By ~ 17

Author(s):

Ewan A. Gibb ◽

David R. Edgell

Keyword(s):

Ribonucleotide Reductase ◽

Large Subunit ◽

Homing Endonuclease ◽

Insertion Site ◽

Gene Organization ◽

Rnase E ◽

Stem Loop ◽

Stem Loop Structure ◽

Expression Of Genes ◽

A Genome

ABSTRACT Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.

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Identification of Ribonucleotide Reductase Protein R1 as an Activator of Microtubule Nucleation in Xenopus Egg Mitotic Extracts

Molecular Biology of the Cell ◽

10.1091/mbc.11.12.4173 ◽

2000 ◽

Vol 11 (12) ◽

pp. 4173-4187 ◽

Cited By ~ 8

Author(s):

Saeko Takada ◽

Takehiko Shibata ◽

Yasushi Hiraoka ◽

Hirohisa Masuda

Keyword(s):

Ribonucleotide Reductase ◽

Large Subunit ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Nucleation ◽

Pole Body ◽

Recombinant Mouse ◽

Xenopus Egg ◽

Essential Subunit

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared fromXenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopussperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to γ- and α/β-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

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