Monophosphate Residue Research Articles

Computational Study on the Electrostatic Interactions between Uracil‐DNA Glycosylase (UDG) and DNA

In DNA repair mechanism, Uracil‐DNA glycosylase (UDG) is one of the most important enzymes that are involved in the process of base excision repair (BER), the function of UDG is to repair the uracil‐induced DNA lesion by removing uracil from damaged DNA. Uracil in DNA may occur due to the cytosine deamination or deoxy uridine monophosphate (dUMP) residues misincorporation during DNA synthesis. Medical evidences haven shown that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, liver cancer, etc. Therefore, the research of UDG is crucial in cancer treatment and prevention, as well as other clinical activities. Here we applied multiple computational methods to study the UDG in several perspectives: Understand the stability of UDG enzyme in different pH conditions; study the differences in charge distribution between the pocket side and non‐pocket side of UDG; analyze the field line distribution at the interfacial area between UDG and DNA; perform electrostatic binding forces analyses of special region of UDG (pocket area) and target DNA base (uracil); as well as investigate the charged residues on UDG binding pocket and binding interface. Our results show that it is the whole UDG binding interface rather than the UDG binding pocket area alone who provides the binding attractive force to a damaged DNA at uracil base.

MODOMICS: a database of RNA modification pathways. 2021 update.

The MODOMICS database has been, since 2006, a manually curated and centralized resource, storing and distributing comprehensive information about modified ribonucleosides. Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5′-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

Computational Study on DNA Repair: The Roles of Electrostatic Interactions Between Uracil-DNA Glycosylase (UDG) and DNA.

Uracil-DNA glycosylase (UDG) is one of the most important base excision repair (BER) enzymes involved in the repair of uracil-induced DNA lesion by removing uracil from the damaged DNA. Uracil in DNA may occur due to cytosine deamination or deoxy uridine monophosphate (dUMP) residue misincorporation during DNA synthesis. Medical evidences show that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, and liver cancer. Therefore, the research of UDG is crucial in cancer treatment and prevention as well as other clinical activities. Here we applied multiple computational methods to study UDG in several perspectives: Understanding the stability of the UDG enzyme in different pH conditions; studying the differences in charge distribution between the pocket side and non-pocket side of UDG; analyzing the field line distribution at the interfacial area between UDG and DNA; and performing electrostatic binding force analyses of the special region of UDG (pocket area) and the target DNA base (uracil) as well as investigating the charged residues on the UDG binding pocket and binding interface. Our results show that the whole UDG binding interface, and not the UDG binding pocket area alone, provides the binding attractive force to the damaged DNA at the uracil base.

Bacterial Polyphosphates Modulate Macrophage Responses to Infection

Abstract Polyphosphates are linear polymers of inorganic monophosphate residues (Pi) and these ancient metabolites exist in all living cells. In bacteria, polyphosphate kinases (PPK) and polyphosphatases (PPX) control the accumulation of polyphosphates from ATP as long chains (>300–1000 Pi) for energy storage and prokaryotic homeostasis. Here, we demonstrate that bacterial (long-chain) polyphosphates bound and were internalized by mouse macrophages. Long-chain polyphosphates modulated the expression of more than 1,800 LPS/TLR4-regulated genes in macrophages. This interference by polyphosphates included antagonism of hundreds of interferon-regulated genes as explained by reduced interferon-β release and responsiveness, reduced phosphorylation of STAT1 and diminished major histocompatibility complex II. Polyphosphates also prevented the LPS/TLR4-induced induction of NOS2 and M1 polarization. In C57BL/6J mice infected with live E. coli, PPK deficiency or polyphosphate neutralization using recombinant PPX resulted in better survival rates, amplified macrophage recruitment and improved pathogen clearance. In conclusion, bacterial polyphosphate metabolites interfere with macrophage responses and the manipulation of such interactions could help to restore immunity to infection.

Avoidance of ribonucleotide-induced mutations by RNase H2 and Srs2-Exo1 mechanisms

Srs2 helicase is known to dismantle nucleofilaments of the Rad51 recombinase to prevent spurious recombination events1–3, 4, 5, 6 and unwind trinucleotide sequences that are prone to hairpin formation7. Here we document a new, unexpected genome maintenance role of Srs2 in the suppression of mutations arising from misinsertion of rNMPs during DNA replication. In cells lacking RNaseH2, Srs2 unwinds DNA from the 5′ side of a nick generated by DNA topoisomerase I8 at a rNMP residue. In addition, Srs2 interacts with and enhances the activity of the nuclease Exo1, to generate a DNA gap in preparation for repair. Srs2-Exo1 thus functions in a novel pathway of nick processing-gap filling that mediates tolerance of rNMPs in the genome. Our results have implications for understanding the basis of Aicardi-Goutieres Syndrome, which stems from inactivation of the human RNaseH2 complex9.

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

The Human Mitochondrial Transcriptome

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

Modification of Lipopolysaccharide with Colanic Acid (M-antigen) Repeats in Escherichia coli

Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides, which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS.MLPS biosynthesis is dependent upon (i) CA induction, (ii) LPS core biosynthesis, and (iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides.

Genetic and structural characterization of the core region of the lipopolysaccharide from Serratia marcescens N28b (serovar O4).

The gene cluster (waa) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [L,D-HeppIIIalpha(1-->7)-L,D-HeppIIalpha(1-->3)-L,D-HeppIalpha(1-->5)-KdopI(4<--2)alphaKdopII] (L,D-Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae, indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an O-antigen-deficient mutant of S. marcescens N28b as well as complementation analysis led to the following proposed structure: beta-Glc-(1-->6)-alpha-Glc-(1-->4))-alpha-D-GlcN-(1-->4)-alpha-D-GalA-[(2<--1)-alpha-D,D-Hep-(2<--1)-alpha-Hep]-(1-->3)-alpha-L,D-Hep[(7<--1)-alpha-L,D-Hep]-(1-->3)-alpha-L,D-Hep-[(4<--1)-beta-D-Glc]-(1-->5)-Kdo. The D configuration of the beta-Glc, alpha-GclN, and alpha-GalA residues was deduced from genetic data and thus is tentative. Furthermore, other oligosaccharides were identified by ion cyclotron resonance-Fourier-transformed electrospray ionization mass spectrometry, which presumably contained in addition one residue of D-glycero-D-talo-oct-2-ulosonic acid (Ko) or of a hexuronic acid. Several ions were identified that differed from others by a mass of +80 Da, suggesting a nonstoichiometric substitution by a monophosphate residue. However, none of these molecular species could be isolated in substantial amounts and structurally analyzed. On the basis of the structure shown above and the analysis of nonpolar mutants, functions are suggested for the genes involved in core biosynthesis.

Oligonucleotides incorporating 7-(aminoalkynyl)-7-deaza-2'-deoxyguanosines: duplex stability and phosphodiester hydrolysis by exonucleases.

Oligonucleotides containing 7-(omega-aminoalkyn-1-yl)-7-deaza-2'-deoxyguanosines (1a-c) were investigated regarding their thermal stability (T(m) values) as well as their phosphodiester hydrolysis catalyzed by exonucleases. Those derivatives are suitable for the labeling of nucleic acid constituents as well as for the postlabeling of DNA. For this, the phosphoramidites 7a,c (obtained from the nucleoside 1a,b), protected by an isobutyryl group at the 2-amino group and a phthaloyl residue at the side-chain amino function, were synthesized. Using compounds 7a,c together with the phosphoramidite of 1c in solid-phase synthesis, a series of self-complementary and non-self-complementary oligonucleotides were prepared and characterized by MALDI-TOF mass spectrometry. A comparison of the T(m) values of the modified oligomers shows that the thermal stability of the duplexes decreases with the length of the nucleobase 7-(omega-aminoalkyn-1-yl) side chain. Exonucleolytic cleavage of oligonucleotide single strands incorporating either the 7-(3-aminopropyn-1-yl)- or the 7-(4-aminobutyn-1-yl)-substituted nucleosides 1a or 1b, respectively, reveals that 3' --> 5' specific snake venom phosphodiesterase liberates 1a 5'-monophosphate but not the methylene-extended 1b 5'-monophosphate. On the contrary, the 5' --> 3' specific bovine spleen exonuclease is able to cleave off single 1a and 1b 3'-monophosphate residues; its action is, however, terminated in the case of oligonucleotides containing two consecutive 1a or 1b nucleotide units.

Efficient sialyltransferase inhibitors based on glycosides of N-acetylglucosamine.

D-Glucosamine was transformed into phenyl and 2-benzoyloxyethyl N-acetylglucosamine beta-glycosides 6a and 6b, respectively. Transformation of 6a,b into 6-O-unprotected N-acetylglucosamine derivatives 9a,b permitted the generation of an aldehyde group in the 6-position. Treatment of these intermediates with base afforded unsaturated aldehyde derivatives 10a,b, which are structural mimics of 2,3-dehydroneuraminic acid. H-Phosphonate addition to the aldehyde group and attachment of the cytidine monophosphate residue to the generated hydroxy group gave fully protected transition state analogues of cytidine monophosphate-N-acetylneuraminic acid 14a,b. Liberation of the unprotected compounds 1ah,l and 1bh,l led to excellent inhibitors of alpha(2-6)-sialyltransferase from rat liver. Variation of the protective group cleavage procedure for 14a,b led to formal loss of phosphate, thus resulting in diene derivatives (E)-/(Z)-2a,b, which also exhibited inhibitory properties.

Mammalian Abasic Site Base Excision Repair: IDENTIFICATION OF THE REACTION SEQUENCE AND RATE-DETERMINING STEPS

Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, beta-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' "nicking" of the AP site-containing DNA strand by AP endonuclease, beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase I to seal the resulting nick. Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system. Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of beta-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.

The first occurrence of polyhydroxylated steroids with phosphate conjugation from the starfish tremaster novaecaledoniae

Abstract Three steroid constituents have been isolated from the starfish Tremaster novaecaledoniae (Jangoux 1982) collected at a depth of 530 m off New Caledonia. Compounds 1 – 3 , designated as tremasterol A – C, are characterized by the presence of 3β-O-sulphated, 6α-O-phosphated and 16β-O-acetylated groupings on a steroidal skeleton. In compound 1 the monophosphate residue is further linked to 1′-glucose (1′-glucose tetracetate in 2 and 1′-glucose-6′-acetate in 3 ).

Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography.

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.

In vitro correction of G.T mispairs to G.C pairs in nuclear extracts from human cells.

In differentiated cells, only a specific subset of genes is expressed. Recently, several genes have been shown to be transcriptionally inactivated by methylation of cytosine residues, mainly within their promoter sequences. Spontaneous hydrolytic deamination of 5-methylcytosine to thymine, which has been estimated to generate up to 12 G.T mismatched base pairs in the human genome per day, could have a deleterious effect on the expression of such genes. We recently reported that mammalian cells possess a specific repair pathway, which counteracts the mutagenic effects of this deamination by correcting G.T mismatches almost exclusively to G.C pairs. We show here that, in nuclear extracts from HeLa cells, this repair is mediated by excision of the aberrant thymidine monophosphate residue, followed by gap-filling to generate a G.C pair. We also provide preliminary evidence that the initial step of this process involves a DNA glycosylase.

Biosynthesis of Lipopolysaccharide in Escherichia coli: Cytoplasmic Enzymes that Attach 3-deoxy-D-manno-octulosonic Acid to Lipid A

Abstract Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.

Human hepatitis delta virus RNA subfragments contain an autocleavage activity.

Hepatitis delta virus (HDV) contains a single-stranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 microM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known self-cleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.

A Family of Glycoinositol Phospholipids from Leishmania major: Isolation, characterization, and antigenicity

The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1–6) by gas chromatographymass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4–6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1–4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18–26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and −6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1–4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and −6), respectively. GIPL-2 and −3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.

Synthesis of a fixed-length single-stranded DNA probe by blocking primer extension in bacteriophage M13

A simple and efficient technique has been developed for preparing radiolabeled single-stranded (ss) probes of determined length and high specific radioactivity. The human beta-globin gene intervening segment II (IVSII) fragment (0.9-kb) was inserted between the EcoRI and BamHI sites of M13mp11 and used as a template for ss probe synthesis. The M13 hybridization probe primer (M13 Hpp) was annealed to the recombinant M13mp11-beta IVSII template DNA. This M13 Hpp was next blocked by the enzymatic addition of a dideoxy adenosine monophosphate (ddAMP) residue to the 3' OH group of the primer. The M13 universal sequencing primer was then annealed and used to prepare an ss copy of the beta-IVSII fragment. Synthesis of the ss fragment was terminated by the presence of the dd-blocked M13 Hpp yielding a specific 0.9-kb ss beta-IVSII probe.