Elsevier

DNA Repair

Volume 11, Issue 5, 1 May 2012, Pages 480-487
DNA Repair

Novel mutator mutants of E. coli nrdAB ribonucleotide reductase: Insight into allosteric regulation and control of mutation rates

https://doi.org/10.1016/j.dnarep.2012.02.001Get rights and content

Abstract

Ribonucleotide reductase (RNR) is the enzyme critically responsible for the production of the 5′-deoxynucleoside-triphosphates (dNTPs), the direct precursors for DNA synthesis. The dNTP levels are tightly controlled to permit high efficiency and fidelity of DNA synthesis. Much of this control occurs at the level of the RNR by feedback processes, but a detailed understanding of these mechanisms is still lacking. Using a genetic approach in the bacterium Escherichia coli, a paradigm for the class Ia RNRs, we isolated 23 novel RNR mutants displaying elevated mutation rates along with altered dNTP levels. The responsible amino-acid substitutions in RNR reside in three different regions: (i) the (d)ATP-binding activity domain, (ii) a novel region in the small subunit adjacent to the activity domain, and (iii) the dNTP-binding specificity site, several of which are associated with different dNTP pool alterations and different mutational outcomes. These mutants provide new insight into the precise mechanisms by which RNR is regulated and how dNTP pool disturbances resulting from defects in RNR can lead to increased mutation.

Highlights

Mutator mutants of E. coli are described with altered ribonucleotide reductase (RNR). ► RNR mutants display altered dNTP pools and corresponding mutational specificities. ► Mutants are found at the enzyme's allosteric activity and specificity sites. ► A novel regulatory important region is discovered in R2 subunit of RNR.

Introduction

The proper control of the intracellular deoxynucleoside-5′-triphosphates (dNTPs) – the direct precursors for DNA synthesis – is critically important for the efficiency and fidelity of DNA replication and the DNA repair processes needed for genomic stability [1]. Genetically and pharmacologically-induced dNTP pool changes have long been recognized to have genotoxic consequences that can lead to mutagenesis and cell death [2], [3] and are also implicated in disease [4], [5], [6], [7].

The controlled production of dNTPs depends on an enzyme termed ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to the corresponding 2′-deoxynucleotides [8]. This reduction is a chemically difficult reaction [9], [10] and requires the presence of a stable organic radical [9], [11], [12]. Based on the precise type of radical, RNRs have been divided into a number of classes [13], [14]. The mammalian RNR and that of many bacteria, yeast, and viruses contain a tyrosyl radical, and this group of enzymes is referred to as class I RNR. Within this class there is extensive conservation of mechanism and structure [12], and the enzyme from Escherichia coli serves as an important model system [12], [15]. The E. coli enzyme, like the mammalian enzyme, reduces nucleoside diphosphates (NDPs) to the corresponding deoxynucleoside diphosphates (dNDPs) [16]. The quaternary organization of this class of RNR is α2β2, in which the large α (or R1) subunits contain the catalytic site and two allosteric effector-binding sites, and the small β (or R2) subunits contain the tyrosyl radical and a dinuclear iron center, both of which are essential for the enzymatic activity [9], [11]. In E. coli, the R1 and R2 subunits are encoded, respectively, by the nrdA and nrdB genes, which form an operon near 50′ on the E. coli chromosome.

RNR regulation occurs at several levels, including transcriptional during the cell cycle [17], [18], [19], [20]. During DNA replication, regulation occurs by intricate allosteric changes, and changes in RNR oligomerization state [21], [22] ensuring adequate levels of the four dNTPs in proper ratios. These allosteric modes of regulation, first described many years ago, are still a subject of intense research [15], [23], [24], [25], [26]. One mode of regulation is an on–off switch governed by the ATP/dATP ratio. The site for the type of regulation, the activity site, is located at the N-terminus of the R1 subunit. By monitoring the ATP/dATP ratio, it assures an overall dNTP level that is presumably optimal for efficient DNA replication. A second regulatory site on R1, named the specificity site, is a binding site for dATP, ATP, dGTP, and dTTP. Depending on which dNTP is bound, the nearby catalytic site is conformationally primed to reduce one specific NDP substrate (ADP, CDP, GDP, or UDP). In this manner, this site regulates the specificity of the enzyme such that the four dNTPs are maintained at their desired ratios.

While significant effort has been expended on studying RNR and its regulation, many of the mechanistic details remain to be understood. Also, few studies have been performed addressing how regulation of RNR affects the dNTP pools in vivo and the fidelity of the DNA replication process [27]. One major limitation to these studies has been the overall lack of RNR mutants with altered dNTP pools. In the present work, we use a new genetic system for obtaining E. coli RNR mutants with a mutator phenotype (i.e., elevated mutation rate). In total, we obtained 23 novel single point mutants with a mutator phenotype resulting from altered dNTP pools. These mutants provide new details regarding the precise modes of RNR regulation and the correlations between dNTP pool changes and mutation rates.

Section snippets

Strains and media

The E. coli strains used are derivatives of strain NR12470, a Δ(gpt-lac)5 derivative of strain MG1655 [28]. The nrdAB-carrying plasmids pHABcat or pHABamp (see description below) were introduced by transformation, after which the chromosomal nrdAB operon was replaced by the Δ(nrdAB::kan) deletion by P1 transduction. The Δ(nrdAB::kan) allele will be described elsewhere (M. Hung and R.M.S.). The strains were then made recA56, srl-360::Tn10 by P1 transduction, followed by introduction of the

Results

The critical role of RNR in furnishing and regulating the cellular dNTPs is well recognized. As noted in Section 1, at least two major RNR feedback mechanisms have been established to operate for maintaining the dNTP pools at both an appropriate level and the desired ratios among the four nucleotides. However, the precise details of these regulatory steps are still being explored. To obtain new insights into these regulatory processes, we have undertaken an approach of directly isolating E. coli

Discussion

Ribonucleotide reductase (RNR) is a crucial enzyme for DNA synthesis as it serves to provide a controlled supply of dNTPs to the DNA synthesis machinery. Despite this importance, relatively little is known about the precise extent to which RNR controls the cellular mutation rate. Our present results identifying a large, new set of E. coli mutator strains confirm the important control that RNR exerts on the cellular mutation rate and provide novel insights into the RNR feedback regulatory

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Drs. W. Copeland, M. Resnick, and S. Wilson of the NIEHS for their helpful comments on the manuscript for this paper and Dr. J. Stubbe and her laboratory for the coordinates of the Uhlin and Eklund model. This work was supported by project Z01 ES065086 of the Intramural Research Program of the National Institute of Environmental Health Sciences (NIEHS).

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