Author Correction: Characterization of a second class Ie ribonucleotide reductase

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of Escherichia coli where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction. Editor's evaluation Nearly all organisms require a ribonucleotide reductase (RNR) to convert ribonucleotides to their deoxyribonucleotide counterparts. In this important study, the reader learns how the model organism Escherichia coli can adapt to survive without any of its three RNRs. Compelling microbiology experiments to develop this model and analysis of compensatory mutations reveals patterns that are conserved in the few known pathogens that have also eliminated their dependence on an RNR. The manuscript will be of interest to microbiologists, biochemists, and those who work on the evolution of microbial metabolism. https://doi.org/10.7554/eLife.83845.sa0 Decision letter Reviews on Sciety eLife's review process Introduction All life on our planet requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides, from ribonucleotides (Nordlund and Reichard, 2006; Lundin et al., 2009). The emergence of ribonucleotide reduction likely drove the transition from RNA to DNA early in the evolution of life (Lundin et al., 2015; Reichard, 1993; Torrents et al., 2002; Poole et al., 2002) and its centrality is reflected in its near ubiquity in organismal genomes (Lundin et al., 2009). Structural analyses indicate that the catalytic cores of ribonucleotide reductases (RNRs) are homologous (Lundin et al., 2015; Sintchak et al., 2002), despite sequence similarities between them being extremely limited (Tauer and Benner, 1997). RNRs catalyse deoxyribonucleotide synthesis via a common mechanism wherein a cysteinyl-free radical is generated in the active site. While this similarity in catalysis underscores a common evolutionary origin, RNRs have diverse mechanisms for radical generation (Nordlund and Reichard, 2006). Three broad, evolutionarily distinct, families exist which are also divided on the basis of their radical generation mechanism (Nordlund and Reichard, 2006; Lundin et al., 2015; Lundin et al., 2010). This divergence likely coincides with adaptation to varied environments (Lundin et al., 2010; Hofer et al., 2012). Class I enzymes are strictly aerobic in that they require oxygen or superoxide for activation (Rose et al., 2018). This typically, though not exclusively (Blaesi et al., 2018; Srinivas et al., 2018), occurs through oxidation of a dimetal ion centre from which a stable cysteine radical is generated, either directly, or indirectly through formation of a tyrosyl radical in the small subunit. Class III enzymes generate a stable glycine radical via cleavage of S-adenosyl methionine, and are strictly anaerobic, while class II enzymes generate a 5'-deoxyadenosyl radical through cleavage of adenosylcobalamin and operate irrespective of oxygen presence or absence. To date, very few species have dispensed with ribonucleotide reduction, albeit indirectly, in that they instead rely on their hosts for deoxyribonucleotides (Lundin et al., 2009). Examples include a strain of Buchnera aphidicola str. Cc from the Cedar bark aphid, Cinara cedri (Pérez-Brocal et al., 2006). B. aphidicola are maternally inherited aphid endosymbionts that synthesise amino acids in short supply in the aphid diet, are obligately intracellular, and appear to be on the path to transitioning to an organelle (Andersson, 2000; Tamas et al., 2002). Two bacterial pathogens that live in very close association with their hosts—Ureaplasma urealyticum (Glass et al., 2000) and Borrelia burgdorferi (Fraser et al., 1997)—have also lost genes for ribonucleotide reduction, as have two eukaryotic parasites, Giardia lamblia and Entamoeba histolytica (Lundin et al., 2009). While ribonucleotide reduction is essential, we reasoned that it should be possible to dispense with genes for ribonucleotide reduction if deoxyribonucleotides or their precursors are available via growth media. We report the successful elimination of all three RNR operons from the bacterium Escherichia coli. The knockout strain was created by introducing a broad-spectrum deoxyribonucleoside kinase from Mycoplasma mycoides (Wang et al., 2001), which we predicted would enable use of media-supplied deoxyribonucleosides for deoxyribonucleotide synthesis in the absence of ribonucleotide reduction. As predicted, the resulting strain is dependent on deoxyribonucleosides in the growth medium but cannot grow if the media are supplemented with deoxyribose (dR) plus the four nucleobases (A, G, C, T). To understand the impact of limited deoxyribonucleoside availability, we subjected our knockout line to experimental evolution followed by genome sequencing. We were interested in establishing whether, under such conditions, our lines would utilise the reverse deoxyriboaldolase (DERA) pathway for deoxyribonucleotide synthesis. This pathway is reversible in vitro (Horinouchi et al., 2006a; Ogawa et al., 2003), and has long been considered a plausible alternative to ribonucleotide reduction (Racker, 1951; Racker, 1952) for dNTP synthesis, with some considering it to be a plausible ancestral route for the origin of DNA (Benner et al., 1989; Poole et al., 2014). Our evolution experiments reveal that this pathway is not coopted for deoxyribonucleotide synthesis following loss of ribonucleotide reduction, with cells grown under limiting deoxyribonucleoside levels exhibiting a filamentous morphology indicative of stress. Instead, it appears that the DERA pathway is a liability under conditions of limited deoxyribonucleotide availability; we observe loss-of-function mutations in deoB, which are predicted to prevent catabolism of 2-deoxy-D-ribose-1-phosphate. Available genome sequence data from obligate intracellular bacteria that lack ribonucleotide reduction indicate that deoB has in fact been lost on multiple occasions, suggesting that recycling of dR is disadvantageous under conditions where this sugar is in limited supply; deletion of deoB would enable this sugar to be rerouted for deoxyribonucleotide synthesis but would also preclude de novo synthesis via the reverse DERA reaction. Our results thus illuminate a key adaptive step taken by obligate intracellular and pathogenic species to mitigate the loss of ribonucleotide reduction. Results Creation of an E. coli line lacking ribonuclease reduction E. coli carries genes for three RNRs: aerobic class Ia (encoded by the nrdAB operon), class Ib (nrdHIEF), and anaerobic class III (nrdDG) (Figure 1). Under aerobic growth, ribonucleotide reduction is primarily performed by the iron-dependent class Ia enzyme (Monje-Casas et al., 2001), while class Ib has a manganese metal centre (Cotruvo and Stubbe, 2011) and is therefore able to support growth when iron is scarce (Martin and Imlay, 2011). Figure 1 with 2 supplements see all Download asset Open asset Deletion of ribonucleotide reductase (RNR) genes in E. coli. (A) Schematic of each of the three RNR operons in E. coli and the genomic regions following scarless deletion. Arrows indicate locations of PCR primers (Supplementary file 1B) used to confirm RNR operon presence/absence. (B) Results of PCR with primers external to all three nrd operons in ΔRNR compared with the wild-type progenitor strain (REL606). Lane 1, ladder; lanes 2 and 6, no template. Lanes 3–5, band sizes consistent with operon deletion (ΔnrdAB 1645 bp, ΔnrdDG 1620 bp and ΔnrdHIEF 1477 bp) in the ΔRNR line. Lanes 6–9, band sizes for all three operons in wild-type E. coli REL606 (nrdAB 5303 bp, nrdDG 4382 bp, and nrdHIEF 5191 bp). Figure 1—source data 1 This file contains unlabelled and labelled full gel pictures for Figure 1. https://cdn.elifesciences.org/articles/83845/elife-83845-fig1-data1-v2.zip Download elife-83845-fig1-data1-v2.zip We reasoned that, while ribonucleotide reduction is an essential process, it should be possible to compensate for its loss via media supplementation. To eliminate ribonucleotide reduction, we sequentially deleted the RNR operons using a scarless deletion protocol (Materials and methods). We first deleted nrdDG, as this operon is known not to be expressed under aerobic conditions. We next deleted the nrdHIEF operon from the resulting ΔnrdDG line. Finally, we attempted to delete the nrdAB operon the ΔnrdHIEF ΔnrdDG line, grown in the presence of deoxyribonucleosides (dNs). We were initially unsuccessful in deleting nrdAB. This is not unexpected as, while dNs are known to be taken up, some (deoxyguanine [dG], deoxycytidine [dC], deoxyadenine [dA]) are unavailable for DNA synthesis, even when genes in the DERA pathway are mutated, preventing their catabolism (Karlström, 1970). We reasoned that may be because of the absence of a suitable deoxynucleoside kinase activity for converting those dNs to deoxyribonucleotides. We therefore tried knocking out nrdAB in the presence of a heterologously expressed deoxyadenosine kinase (dAK) gene from M. mycoides. Mycoplasmas are known to possess deoxyadenosine kinases genes that permit utilisation of dNs for DNA synthesis (Wang et al., 2001). To establish if resulting lines lacked all three nrd operons, we first PCR screened for evidence of chromosomal nrd operon deletions. Our results indicate that all three operons were successfully deleted (Figure 1). As deletion does not exclude relocation of functional gene copies to another genomic location, we confirmed gene absence via PCR using primers internal to nrd genes and performed RT-PCR to confirm absence of gene expression (Figure 1—figure supplement 1). Finally, knockout status was confirmed with whole genome sequencing. This confirmed deletion of all genes for ribonucleotide reduction, with the nrdAB operon successfully deleted under heterologous expression of dAK from M. mycoides (Figure 1—figure supplement 2). Of four knockout lines, we selected one isolate (hereafter called ΔRNR) for all subsequent work. In addition to lacking all nrd genes, genome sequencing revealed 23 SNPs that likely appeared during the creation of ΔRNR from wild-type progenitor line (REL606) (Supplementary file 1C). ΔRNR is dependent on dN supplementation Our ΔRNR line was created in the presence of dN supplementation, but this does not mean it is dependent on dN supplementation for growth. We therefore sought to understand what supplementation, if any, our ΔRNR line requires. To determine the lowest concentration of dNs that permits growth, we generated a series of growth curves for differing dN concentrations. At high dN concentrations (1 mg/mL), ΔRNR grows favourably, though shows a clear lag compared to wild-type on equivalent media (Figure 2A). At lower dN concentrations, ΔRNR growth is impaired, but there is still discernible growth at 0.05 mg/mL (Figure 2C). When this is dropped to 0.01 mg/mL we observed only marginal growth of ΔRNR (Figure 2D). As expected, in the absence of dN supplementation no growth is observed, while wild-type lines are unaffected (Figure 2E). Finally, we tried growing ΔRNR on dR plus the four bases (A, G, C, T). No growth was observed (Figure 2F). These results indicate that deletion of the three nrd operons from ΔRNR has completely eliminated the capacity for de novo deoxyribonucleotide synthesis; no other genes appear able to compensate for this deficiency. Figure 2 Download asset Open asset ΔRNR shows growth across a range deoxyribonucleoside (dN) concentrations but cannot grow in the absence of dN supplementation. Growth was monitored for 25 hr in 1× MOPS media + 1% glucose with supplementation, as indicated. Wild-type (REL606, black), ΔRNR (orange). Curves show mean OD595, error bars show SEM, all experiments performed in triplicate. (A) 1 mg/mL, (B) 0.25 mg/mL, (C) 0.05 mg/mL, (D) 0.01 mg/mL, and (E) 0 mg/mL dNs. (F) 0.1 mg/mL deoxyribose (dR) + 0.25 mg/mL of adenine (A), cytosine (C), thymine (T), and guanine (G). We next sought to establish whether ΔRNR requires supplementation of all four dNs, or whether any are dispensable. Strains were once again grown in 1× MOPS + 1% glucose, with individual dNs (dA, dG, deoxythymine [dT], or dC) added at a concentration of 0.25 mg/mL. This revealed that ΔRNR is unable to grow in the presence of dA, dG, or dT (either alone [Figure 3A–C] or in combination [Figure 3E]), even after 44 hr of monitoring (data not shown). However, ΔRNR can grow on dC alone (Figure 3D). Together these data indicate dC is the sole dN required for ΔRNR growth in minimal media. Figure 3 Download asset Open asset ΔRNR requires only deoxycytidine (dC) for growth in minimal media. Growth was monitored for wild-type (REL606, black) and ΔRNR (orange) in 1× MOPS media + 1% glucose, with deoxyribonucleoside (dN) supplementation, as indicated. Growth was monitored for 25 hr. Curves show mean OD595, error bars show SEM, all experiments performed in triplicate. (A) Deoxyadenine (dA) (0.25 mg/mL), (B) deoxyguanine (dG) (0.25 mg/mL), (C) deoxythymine (dT) (0.25 mg/mL), (D) deoxycytidine (dC) (0.25 mg/mL), (E) dA, dG, dT, each at 0.25 mg/mL. ΔRNR exhibits a filamentous cell morphology when grown under limiting dNs During our growth assays we observed a 'clumping' phenotype when ΔRNR is grown in liquid media at concentrations of dNs that limit growth. This contrasts with the uniform cloudy appearance of wild-type E. coli (Figure 4C, top left panel). Examination of cells at ×100 magnification revealed that ΔRNR cells are elongated and filamentous at very low levels of dNs, whereas ΔRNR cells have a similar morphology to wild-type at higher dN concentrations (Figure 4A). Some cells reached lengths several times that of wild-type (Supplementary file 1D). Figure 4 Download asset Open asset ΔRNR exhibit a reversible elongated, filamentous cell morphology with multiple nucleoids when grown in limiting deoxyribonucleoside (dN) concentrations. (A) ×100 Brightfield photographs of REL606 and ∆RNR at 1 and 0.01 mg/mL. (B) FITC and DAPI staining at ×100 of REL606 and ∆RNR at 0.01 mg/mL. (C) ∆RNR phenotype is reversible if dN concentration is increased. Loss of clumpy phenotype occurs when ∆RNR cells are transferred from low (0.01 mg/mL) to high (1 mg/mL) dNs. Brightfield microscopy images reveal that this change is accompanied by a reduction in elongated cell morphology upon transfer to high [dN] (magnification ×1000; scale bar 20 μM). This phenotype appears most pronounced at low dN concentrations. We therefore sought to establish if this phenotype is the result of environmental conditions. We transferred a population of ΔRNR cells exhibiting the filamentous cell phenotype from low dN (0.01 mg/mL) media to a less restrictive environment ([dN]=1 mg/mL). Following transfer to higher [dN], the filamentous phenotype is heavily diminished (Figure 4C). This demonstrates that the phenotype is environmental and can be reversed if cells are moved to a high [dN] environment. One possibility is that, under low [dN], cells are growing but unable to complete cell division. If so, this might be reflected by the presence of multiple DNA-dense regions across the length of the cells. To visualise DNA within filamentous cells, we stained ΔRNR cells grown in low [dN] (0.01 mg/mL) with FITC and DAPI. This revealed the presence of multiple DAPI-stained regions across the length of the cells (Figure 4B), suggestive of the presence of multiple DNA nucleoids in ΔRNR cells grown at low [dN]. Evolution of ∆RNR lines under restricted dN supplementation The loss of ribonucleotide reduction in obligate intracellular lifestyles presumably resulted from relaxed selection on deoxyribonucleotide production when deoxyribonucleotides are available from the environment. However, for both parasitism and endosymbiosis, the host dNTP pool must be shared. We were therefore interested to assess if the ∆RNR line adapts to a reduction in dN availability, as might occur in the evolutionary switch from de novo synthesis to dependency on host production. In order to allow the strains to adapt to a lower concentration of dN supplementation in the growth media, we initiated evolution experiments at dN concentrations where ∆RNR lines grow, but not as well as wild-type (1 mg/mL and 0.25 mg/mL). Five independent lines of ∆RNR were serially passaged in each condition by growing the culture to stationary phase, then transferring 50 μL into a new six-well plate containing 5 mL of fresh MOPS media + 1% glucose and dNs (Figure 5). We also established three control lines of the wild-type progenitor strain (REL606) at each dN concentration. The initial experiment was run for a total of 30 transfers. Glycerol stocks were created every five transfers for each line, with contamination checks performed concurrently. Figure 5 Download asset Open asset Overview of the ΔRNR evolution experiment. Five lines of ΔRNR and three lines of wild-type progenitor (REL606) were established at one of two conditions (1 mg/mL or 0.25 mg/mL deoxyribonucleosides [dNs] in MOPS + 1% glucose), and serially passaged for 30 transfers. Genomic material from each line was then extracted from a single colony and sent for sequencing giving us clonal-level genome information. To further investigate adaptation to low concentrations of dNs, the 'fittest' ΔRNR line grown at 0.25 mg/mL dNs (ΔRNR_250_T30_1) was used to seed a subsequent experiment. Ten replicate lines of ΔRNR_250_T30 _1 were serially passaged for an additional 10 transfers in MOPS + 1% glucose and 0.01 mg/mL dNs. DNA from a single colony of each of these 10 lines (ΔRNR_10_T40_1–10) was then extracted and sent for whole genome sequencing. Elongate cell morphology in ∆RNR lines diminishes over the course of the evolution experiment Our initial observations of unevolved ∆RNR (henceforth ∆RNR_T0) revealed an elongated filamentous cellular phenotype at lower concentrations of dNs (Figure 4). We therefore monitored cell morphology during our evolution experiment. Every five transfers, we examined length of ∆RNR and REL606 cells from each condition (1 mg/mL or 0.25 mg/mL dNs; cells from well position and or were used for all and mean length At the of the experiment, ∆RNR cells are than at 0.25 mg/mL dNs (Figure We cell lengths of 20 at this lower concentration of a times the length of wild-type cells. the the experiment reached 30 transfers, cell length was to wild-type at both dN concentrations (Figure These in morphology cells to adapt to restricted dN Figure Download asset Open asset The ∆RNR cell phenotype diminishes over the course of an evolution experiment. (A) cell length during the course of a evolution experiment. length was for 20 cells for replicate line 1 of ∆RNR and REL606 in either 1 mg/mL or 0.25 mg/mL deoxyribonucleosides (dNs). were taken every five transfers. Over the course of the evolution experiment, ∆RNR cell length at both dN concentrations. (B) of wild-type and ∆RNR cells at transfer 1 and transfer 30 at each dN concentration (1 mg/mL or 0.25 mg/mL) (magnification ×1000; scale bar 2 μM). ∆RNR lines exhibit growth following evolution under restricted dN availability 30 transfers, we sought to determine if ∆RNR lines their capacity to grow under restricted (either 1 mg/mL or 0.25 mg/mL) dN We use the following for our evolution is replicate line of ∆RNR at 1 mg/mL dNs for 30 transfers. At 1 mg/mL, through all growth to (Figure compared to which no growth at 0.01 mg/mL dNs (Figure all lines growth at this low concentration (Figure similar of was also for the five ∆RNR replicate lines in 0.25 mg/mL (Figure supplement 1). that cell morphology and preclude use of for of or cell et al., in the error to REL606 can only be used to a of growth. All lines to grow in the absence of dN supplementation (Figure Figure supplement they on media supplementation. Figure with 1 supplement see all Download asset Open asset Growth of ∆RNR lines at 1 mg/mL deoxyribonucleosides for 30 transfers. Growth was monitored for wild-type and lines in 1 mg/mL dNs for 30 Growth experiments were performed in 1× MOPS media + 1% glucose with the addition of dNs at concentrations. Growth was monitored for 25 hr. Curves show mean OD595, error bars show SEM, all experiments performed in triplicate. (A) 1 mg/mL dNs, (B) 0.25 mg/mL dNs, (C) mg/mL dNs, (D) mg/mL dNs, (E) 0 mg/mL dNs. Evolution of ∆RNR at 0.25 mg/mL dNs results in Following 30 transfers, we the genomes of our lines in 0.25 mg/mL dNs and 1 mg/mL dNs and each of three wild-type control lines and from single The filamentous phenotype of our ∆RNR lines under restricted dN supplement of the number of which in of We therefore report total observed mutations plus for each experimental line (Figure file Our knockout lines and all mutations than wild-type at the dN concentration (Figure file Our lines the of mutations lines exhibit a small but in total SNPs to wild-type (Figure supplement 1 and file was no in total mutations between wild-type lines in either 1 mg/mL or 0.25 mg/mL dNs Figure with 4 supplements see all Download asset Open asset observed following experimental evolution of ∆RNR for 30 transfers. single plus in each line at evolution in 1 mg/mL deoxyribonucleosides RNR evolution in 0.25 mg/mL, RNR is well that RNRs deoxyribonucleotide pool sizes through et al., 2012). We were therefore interested to see whether loss of ribonucleotide reduction resulted in in E. coli. Our was that there would not be any as our lines are supplemented with concentrations of all four dNs. If there was we this to be most in our lines, as these the number of of SNPs in our lines are of one (Figure Figure supplement 1). ∆RNR lines of our genome data from lines at transfer 30 revealed that one which codes for was in of 10 lines from transfer 30 (Supplementary file and of and dC to and of the data revealed that of lines an deletion (Supplementary file Figure supplement while one line an that was predicted to result in a As the are we that this deletion may have in the ancestral but was not at the we initiated our evolution experiment. These genome data were from colony it was not possible to determine if this was the the of this was in the of our evolution experiment. To determine whether cdd is lost in response to the experimental conditions, we screened our ∆RNR to establish if any cdd. We PCR of stocks for two knockout lines This revealed that the that we used to establish our evolution experiments was a with both and cdd deletion (Figure supplement it appears that, after 30 transfers, all 10 lines were for the deleted this the deletion is very likely to have in the ancestral and that our lines were from a despite the knockout lines being established from individual following scarless deletion of nrdAB and methods). of revealed no evidence of a deletion (Figure supplement We therefore established a short evolution experiment with five independent lines, in 0.25 mg/mL dNs, the early of our evolution experiment with the two transfers, we observed the emergence of a cdd deletion in several replicate lines (Figure supplement In similar experiments not and using these lines, we see loss of the cdd under similar growth conditions (data not shown). Finally, that an in cdd but not the suggesting that it is deletion of the cdd that is selected under these conditions. ∆RNR lines in 0.01 mg/mL dNs the DERA pathway transfer one of our lines growth similar to wild-type when grown in 0.25 mg/mL dNs (Figure supplement 1). this line as we initiated a evolution experiment using 10 lines, grown in 0.01 mg/mL dNs (Figure 5). 10 transfers, we these lines All a of with SNPs (Figure supplement 4). As with the lines from our first 30 transfers, the of mutations were Genome analysis of lines revealed mutations (Supplementary file of which were in than one line. Of these were in two lines, in three lines, and 4 in four lines (Supplementary file One deoB, was in lines and is in the of our experiment in that it codes for This enzyme the transfer of a between the and of and is for the step in deoxyribonucleotide (Figure analysis revealed that several of the mutations to deoB were is possible that

Biochemical reactions, even those as complex as replicating the DNA genome of cells, follow the principle that the process is regulated by both the substrate concentration and by the enzymes that mediate the process. Deoxynucleoside triphosphates (dNTPs), the substrates for DNA polymerizing enzymes, have long been known to be limited in their concentration in cells because the enzyme that synthesizes deoxynucleotides from ribonucleotides, ribonucleotide reductase (RNR), is synthesized and enzymatically activated as cells enter the S phase (1, 2). RNR, discovered by Peter Reichard 52 y ago (3), converts all four ribonucleotide diphosphates (rNDPs) to the respective deoxynucleoside disposphates (dNDPs), which are then rapidly converted to dNTP. Low levels and activity of RNR provide sufficient dNTPs for mitochondrial DNA synthesis and for DNA repair in noncycling cells and during the G1 phase of the cell-division cycle in proliferating cells, but RNR levels and activity are hugely increased as cells commit to replicate DNA during the S phase of the cell-division cycle or following extensive DNA repair (4). Indeed, RNR is one of the most highly regulated enzymes known. The mammalian enzyme synthesizes all four dNDPs in a cycle, is allosterically activated by dATP, dTTP, and dGTP to balance the relative levels of the four dNTPs (dCTP, dTTP, dGTP and dATP), and is feed-back–inhibited by dATP, because dATP is the last dNTP to be made in the cycle of synthesizing all four dNTPs by a single RNR enzyme (1). Specific inhibitory proteins (in yeasts) also control RNR activity and RNR subunit levels are regulated by cell cycle-dependent transcription of the genes encoding the subunits and by subunit protein stability (4, 5). On the basis of these observations, one might expect that dNTP synthesis by RNR should be sufficient to control how and when genome DNA replication occurs because RNR is only maximally active during the S phase. However, recent studies, including those emerging from far-afield studies of how HIV replication is restricted to certain cell types (6, 7), have uncovered a new control of dNTP levels, dNTP destruction. The sterile alpha motif and HD-domain containing protein 1 (SAMHD1) protein is a deoxynucleoside triphosphohydrolase that cleaves dNTPs to the respective deoxynucleoside and a triphosphate (8). In PNAS, Franzolin et al. (9) show that dNTP destruction by SAMHD1 also contributes to dNTP concentration control during the cell-division cycle of proliferating cells, thereby affecting both DNA replication and cell-cycle progression.

Arcobacter spp., such as Arcobacter bivalviorum (A. bivalviorum), are free-living organisms found in diverse environments and associated with animals. They are considered emerging enteropathogens and potential zoonotic agents. Ribonucleotide reductase (RNR) is the key enzyme that is used to convert ribonucleotides into deoxyribonucleoside triphosphates (dNTPs). This process utilises radical-based chemistry and is crucial for DNA biosynthesis and repair. There are three RNR classes, with class I RNR the most studied, present in A. bivalviorum, eukaryotes, and many prokaryotes. Class I RNRs are further divided into three subclasses: Ia, Ib, and Ic. Class Ib RNRs use a dimanganese-oxo centre, unlike class Ia RNRs, which use a diiron-oxo centre. A. bivalviorum possesses a class Ia enzyme that requires a diferric tyrosyl radical cofactor located within its beta (β) subunit. Indeed, both the efficiency and fidelity of DNA synthesis are influenced by the stability of the tyrosyl radical (Y•) in the RNR, which is a critical aspect of its enzymatic function. This study investigates the stability of the Y-radical (Y•) site within the RNR β subunit of A. bivalviorum and the nature of the neighbouring amino acid residues. To achieve these goals, we developed a model of the RNR β subunit of A. bivalviorum, using the RNR β subunit of Aquifex aeolicus as a reference template (7aik.1. A PDB). The results provide some important details about the radical site and its surrounding residues, highlighting the influence of the protein structure on the stability of the radical. These findings may guide the development of novel inhibitors targeting this enzyme in A. bivalviorum.

2066 Background: We evaluated the efficacy and tolerability of gemcitabine monotherapy in heavily pretreated breast cancer patients, and the polymorphism of gemcitabine-related genes to elucidate the association with toxicity. We detected the polymorphism of deoxycytidine kinase (dCK), deoxycytidine deaminase (dCDA), and ribonucleotide reductase (RNR). Methods: A weekly infusion of gemcitabine at 850mg/m2 for 3 of every 4 weeks was introduced in patients who had failed previous doxorubicin and taxane. Treatment was delayed until leukopenia was recovered with G-CSF support without dose modification. The known polymorphism site of 3 genes were evaluated by CEQ™ 8000 Genetic Analysis System (Beckman): dCK(GG->GT), dCDA(AA->AG), RNR(CC-> CT, TT->TC) with genomic DNA of peripheral lymphocytes. Results: Of 51 enrolled patients, 47 were evaluable. Total 208 cycles were administered and the relative dose intensity was 89%. The response rate was 20%. Median response duration and overall survival were 8 and 12 months, respectively. The toxicity was mild with 14% of grade III neutropenia and 6% of grade III/IV thrombocytopenia. Polymorphism was found in 13% of dCK, 11% of dCDA. 42% of the 1st RNR, 38% of the 2nd RNR and 18% of both RNR sites. By Gibbs sampling-based algorithm, AGCT was the most frequent type (0.6226) with 3 genes, and CT was the most common haplotype in RNR (0.6909). When we evaluated the association of the 4 polymorphic sites from 3 genes with maximum leukopenia, AGTC type polymorphism showed the correlation with leukopenia. Moreover, the number of polymorphism in RNR and the polymorphism of the second site in RNR showed the significant correlation with leukopenia. Subsequently, patients with more number of polymorphism in 3 genes or polymorphism in RNR gene required more numbers of G-CSF rescue without gemcitabine dose reduction. Finally, patients with AGCT type showed a less response to treatment. Conclusions: Gemcitabine monotherapy is effective as salvage treatment and dose adjustment can be done effectively by pharmacogenomic association study in heavily pretreated, breast cancer patients. No significant financial relationships to disclose.

Ribonucleoside diphosphate (RDP) reductase activity can be readily assayed in ether-treated Escherichia coli cells. The rate of cytidine 5'-diphosphate (CDP) reduction observed in ether-treated cells by using saturating substrate concentrations is about 25% of the rate of de novo deoxyribonucleotide synthesis required to account for in vivo deoxyribonucleic acid synthesis. Optimal activity is observed in the presence of magnesium ions and a positive effector. Adenosine 5'-triphosphate (ATP), deoxy ATP (dATP), and deoxythimidine triphosphate serve as positive effectors, and dATP also serves as a negative effector. These effects on the activity in ether-treated cells resemble those observed in vitro with highly purified enzyme. When the RDP reductase activity in these cells is assayed by using high specific activity (3)H-CDP as substrate, even at nonsaturating substrate concentrations, the sensitivity of the assay is sufficient to make it useful for the assay of the low levels of reductase activity in cells not derepressed by thymine starvation or in cells containing mutationally altered RDP reductase. This assay is much easier to perform than the usual in vitro assay, since thioredoxin, thioredoxin reductase, and enzyme subunits B1 or B2 need not be first purified and added to the reaction mixtures.

Hsp70 is a well-conserved molecular chaperone involved in the folding, stabilization, and eventual degradation of many “client” proteins. Hsp70 is regulated by a suite of co-chaperone molecules that assist in Hsp70-client interaction and stimulate the intrinsic ATPase activity of Hsp70. While previous studies have shown the anticancer target ribonucleotide reductase (RNR) is a client of Hsp70, the regulatory co-chaperones involved remain to be determined. To identify co-chaperone(s) involved in RNR activity, 28 yeast co-chaperone knockout mutants were screened for sensitivity to the RNR-perturbing agent Hydroxyurea. Ydj1, an important cytoplasmic Hsp70 co-chaperone was identified to be required for growth on HU. Ydj1 bound the RNR subunit Rnr2 and cells lacking Ydj1 showed a destabilized RNR complex. Suggesting broad conservation from yeast to human, HDJ2 binds R2B and regulates RNR stability in human cells. Perturbation of the Ssa1-Ydj1 interaction through mutation or Hsp70-HDJ2 via the small molecule 116-9e compromised RNR function, suggesting chaperone dependence of this novel role. Mammalian cells lacking HDJ2 were significantly more sensitive to RNR inhibiting drugs such as hydroxyurea, gemcitabine and triapine. Taken together, this work suggests a novel anticancer strategy-inhibition of RNR by targeting Hsp70 co-chaperone function.

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4β4 in Escherichia coli , wherein β is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and β2 dimeric motifs within the respective α6 or α4β4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -β to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or β2 associations in the dATP-bound α6 or α4β4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

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

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.

An L1210 cell line (MQ-580) selected for resistance to the ribonucleotide reductase inhibitor, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ), had been previously shown to have altered properties related to the non-heme iron subunit of ribonucleotide reductase (RR). In addition, the MQ-580 cells had other metabolic alterations that were consistent with multidrug resistance. Neither the wild-type (WT) nor the MQ-580 cells showed a G0/G1 block in response to X-irradiation. Since the MQ-580 cells also showed resistance to adriamycin (ADR), the WT cells were more sensitive to the effects of ADR than the MQ-580 cells. However, when the MQ-580 cells were treated with the combination of ADR plus verapamil, the MQ-580 cells showed cellular responses that included cell cycle block in G2/M and increased apoptosis. The WT cells, in response to the combination of ADR plus verapamil, blocked in the S-phase of the cell cycle with an increased necrotic cell population in comparison to treatment with ADR alone. While MAIQ could be shown to cause an apoptotic response in the WT and MQ-580 cells, the concentrations of MAIQ required to induce apoptosis were 20- to 40-times the IC50 value. However, even though the MQ-580 cells were 8-fold more resistant to MAIQ than the WT cells, the MQ-580 cells were more sensitive to MAIQ-induced apoptosis. These data show that alterations at the RR site lead to phenotypic expressions not overtly related to RR. However, because of the critical role that RR plays in cell division and DNA repair, the changes observed with respect to cell cycle and apoptosis, may in fact, be a direct consequence of the alteration at RR.

The effects of prolonged exposure to the ribonucleotide reductase (RR) inhibitor, hydroxyurea (HU), were assessed in the presence or absence of recombinant interferon alfa-2a (IFN) in wild-type human colon cancer cells (HT-29) and variants expressing low-level resistance to HU (R200). IFN at nontoxic concentrations decreased the IC50 of HU from 368 microM to 215 microM (P < 0.01) in wild-type cells, but not in the resistant variants. Potential cellular targets for the HU/IFN interaction were examined. In wild-type, but not resistant cells, treatment with HU at clinically achievable concentrations (1000 microM) resulted in rapid early inhibition of RR activity between 4 and 24 h after treatment with a maximal decrease of 65% at 12 h, decreases in cellular levels of dATP, dCTP and dGTP by 50-90% over the same time course, and a two- to fourfold increase in the level of mRNA for both the M1 and M2 subunits of RR, at 24, but not between 1 and 4 h, which probably represents a response to the earlier decrease in RR activity. IFN at a clinically achievable concentration (500 U/ml) failed to augment the effects of HU on RR protein, RR mRNA levels or RR enzyme activity in either the wild-type or resistant cells, suggesting that the mechanism by which IFN augments the effects of HU in the wild-type cells is independent of the effects of HU on M2.

Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.

&lt;div&gt;Abstract&lt;p&gt;Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR &lt; 0.05) that were more cytotoxic to &lt;i&gt;BAP1&lt;/i&gt;-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (&lt;i&gt;BAP1&lt;/i&gt;&lt;sup&gt;+/−&lt;/sup&gt;) overexpressing &lt;i&gt;BAP1&lt;/i&gt; C91A (catalytically dead mutant) was more resistant to RNR inhibition, while &lt;i&gt;BAP1&lt;/i&gt; knockdown in the &lt;i&gt;BAP1&lt;/i&gt;-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in &lt;i&gt;BAP1&lt;/i&gt;-proficient cell line–derived spheroids compared with &lt;i&gt;BAP1&lt;/i&gt; deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in &lt;i&gt;BAP1&lt;/i&gt; mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with &lt;i&gt;BAP1&lt;/i&gt;-WT but not with &lt;i&gt;BAP1&lt;/i&gt; C91A. Upregulation of RRM2 in NCI-H2452-&lt;i&gt;BAP1&lt;/i&gt; WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.&lt;/p&gt;&lt;/div&gt;

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.