Primer initiation and extension by T7 DNA primase

T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.     

Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis

The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.     

Crystal structure of the C-terminal domain of human DNA primase large subunit

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes’ large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.     

Characterization of DNA primase complex isolated from the archaeon, Thermococcus kodakaraensis

In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3' → 5' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.     

Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.     

Interplay between primase and replication factor C in the hyperthermophilic archaeon Sulfolobus solfataricus

The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes long RNA and DNA products in vitro. How primer synthesis by primase is coupled to primer extension by DNA polymerase in this organism is unclear. Here we show that the small subunit of the clamp loader replication factor C (RFC) of S. solfataricus interacted with both the catalytic and non-catalytic subunits of the primase by yeast two-hybrid and co-immunoprecipitation assays. Further, the primase-RFC interaction was also identified in the cell extract of S. solfataricus. Deletion analysis indicated that the small subunit of RFC interacted strongly with the N-terminal domain of the catalytic subunit of the primase. RFC stimulated dinucleotide formation but decreased the amount of primers synthesized by the primase. The inhibition of primer synthesis is consistent with the observation that RFC reduced the affinity of the primase for DNA templates. On the other hand, primase stimulated the ATPase activity of RFC. These findings suggest that the primase-RFC interaction modulates the activities of both enzymes and therefore may be involved in the regulation of primer synthesis and the transfer of primers to DNA polymerase in Archaea.     

Crystal structure of the C-terminal domain of human DNA primase large subunit: Implications for the mechanism of the primase—polymerase α switch

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes'' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.Key words: DNA primase, prim1, prim2, replication, 4Fe-4S cluster, crystal structure, DNA polymerase α     

Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii

Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.     

Archaeal primase: bridging the gap between RNA and DNA polymerases

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.     

Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product.

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.     

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.     

The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase alpha-primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [gamma-(32)P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase alpha-primase complex.     

Shared Active Site Architecture between the Large Subunit of Eukaryotic Primase and DNA Photolyase

Background

DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood.

Methodology/Principal Findings

Here we report the crystal structure of the yeast PriL-CTD at 1.55 Å resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase.

Conclusion/Significance

Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.     

Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.     

Structural characterization and corepressor binding of the Escherichia coli purine repressor.

The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.     

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.     

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.     

'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.