Rolf Eliasson

Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12–dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.

Cross-talk between the Allosteric Effector-binding Sites in Mouse Ribonucleotide Reductase

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the “activity site” (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resemblesEscherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

Replication of polyoma DNA in isolated nuclei: III. The nucleotide sequence at the RNA—DNA junction of nascent strands

Abstract Short fragments consisting of about 100 to 140 deoxyribonucleotides serve as intermediates in the elongation of polyoma DNA. In nuclei isolated from polyoma-infected 3T6 mouse fibroblasts these fragments are initiated by stretches of RNA. We investigated the nature of the ribo- and deoxyribonucleotides at the RNA-DNA link. DNA was synthesized in vitro from each of the four α-32P-labelled deoxynucleoside triphosphates, the nascent strands were hydrolysed with alkali and the transfer of isotope to ribonucleotides was studied after fractionation of strands according to size. Each strand contained on the average one RNA-DNA link at the 5′ end of DNA. All four common ribo- and deoxyribonucleotides were present at the RNA-DNA link with close to equal frequency, irrespective of chain length or incubation time. In a second approach, daughter strands synthesized in vivo were treated with alkali and the 5′-OH ends of DNA liberated were 32P-labelled using polynucleotide kinase. All four deoxynucleotides were labelled by this treatment confirming the corresponding results of the in vitro experiments. During the discontinuous synthesis of polyoma DNA the switch from RNA to DNA synthesis is thus not effected by a specific sequence at the RNA-DNA junction, in contrast to Escherichia coli where the sequence p(rPy)p(dC)p was reported.

The Active Form of the R2F Protein of Class Ib Ribonucleotide Reductase from Corynebacterium ammoniagenes Is a Diferric Protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducibleEscherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77–95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

The anaerobic (Class III) ribonucleotide reductase from Lactococcus lactis: Catalytic properties and allosteric regulation of the pure enzyme system

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence ofS-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S]+ cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with theL. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Allosteric Control of Three B12-dependent (Class II) Ribonucleotide Reductases IMPLICATIONS FOR THE EVOLUTION OF RIBONUCLEOTIDE REDUCTION

Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii andThermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For theL. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes.

Replication of polyoma DNA in isolated nuclei: II. Evidence for semi-conservative replication

Abstract Nuclei from polyoma-infected mouse fibroblast 3T6 cells catalysed the incorporation of [ 3 H]dTTP into the replicative intermediate of polyoma DNA. The nature of this process was studied by the introduction of a density label into the viral DNA. For this purpose dBrUTP replaced dTTP in the in vitro system and the product formed in the presence of [ 14 C]dATP was analysed by equilibrium centrifugation in neutral CsCl or alkaline Cs 2 SO 4 gradients. The results suggest that the density label was incorporated into newly synthesized progeny strands and that the incorporation process represented semi-conservative replication. In a second type of experiment the replicative intermediate was first pulselabelled in vivo with [ 3 H]thymidine. The isolated nuclei were then incubated in vitro with dBrUTP and [ 14 C]dATP. From an analysis of the distribution of the two isotopes in buoyant density gradients we propose that in vitro synthesis represented elongation of essentially all replicative intermediate molecules present in the nuclei at the time of isolation. From the observed density shifts we could calculate that the initial rate of the in vitro elongation process corresponded to a replication time of about 20 minutes and thus may amount to about 20% of the reported in vivo rate.

Characterization of the RNA initiating the discontinuous synthesis of polyoma DNA

Summary The discontinuous synthesis of polyoma DNA in infected isolated nuclei from 3T6 cells is initiated with RNA. From experiments in which this RNA was labelled from [3H] or β-[32P] labelled ribonucleoside triphosphates we conclude that the 5′ end of initiating RNA starts with ATP or GTP, but not with CTP or UTP. When the RNA was released from progeny DNA strands by digestion with pancreatic DNAse and characterized by gel electrophoresis its position on the gel corresponded approximately to that of a decanucleotide. While initiating RNA was quite homogenous in size it had no unique nucleotide sequence.

Replication of polyoma DNA in isolated nuclei: VII. Initiator RNA synthesis during nucleotide depletion☆☆☆

Abstract Earlier experiments demonstrated that the Okazaki fragments synthesized during discontinuous polyoma DNA synthesis in isolated nuclei at their 5′ ends contained structural elements consisting of polyribonucleotides starting with ATP or GTP (Reichard et al. , 1974) . These structures could be released by digestion with pancreatic DNAase and were named initiator RNA. They consist of a large family of polyribonucleotides differing in base sequence but having a common size of about a decanucleotide. We now demonstrate that limitation of DNA synthesis by low concentrations of deoxyribonucleoside triphosphates in parallel limits the synthesis of initiator RNA. This is additional evidence for the primer function of initiator RNA. When ribonucleoside triphosphates other than ATP were deleted from the incubation medium only a small decrease of DNA and initiator RNA synthesis occurred. Under those conditions deoxyribonucleotides substituted for ribonucleotides and were incorporated internally into the primer. From this result as well as the insensitivity of initiator RNA synthesis to α-amanitin (Reichard & Eliasson, 1979) we suggest that a mammalian counterpart to primase, the dna G gene product of Escherichia coli (Rowen & Kornberg, 1978 a ) , catalyzes the synthesis of initiator RNA.