Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.

Thymidylate synthase-catalyzed, tetrahydrofolate-dependent self-inactivation by 5-FdUMP.

In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Urine 5MedC, a Marker of DNA Methylation, in the Progression of Chronic Kidney Disease.

BACKGROUND: Alterations in DNA methylation may be involved in disease progression in patients with chronic kidney disease (CKD). Recent studies have suggested that 5-methyl-2'-deoxycytidine (5MedC) may be a marker of hypermethylation of DNA. Currently, there is no information available regarding the urine levels of 5MedC and its association with the progression of CKD. METHOD: We examined the urine levels of 5MedC in spot urine samples from 308 patients with CKD (median age: 56 years, male: 53.2%, and glomerulonephritis: 51.0%) using a competitive enzyme-linked immunosorbent assay and investigated the relationships among urine 5MedC, urine albumin, urine α1-microglobulin (α1MG), and the laboratory parameters associated with CKD. The patients were followed for three years to evaluate renal endpoints in a prospective manner. RESULTS: The urine 5MedC level was significantly increased in the later stages of CKD compared to the early to middle stages of CKD. In multiple logistic regression models, urine 5MedC was significantly associated with the prediction of later CKD stages. Urine 5MedC (median value, 65.9 µmol/gCr) was significantly able to predict a 30% decline in the estimated GFR or a development of end-stage renal disease when combined with macroalbuminuria or an increased level of urine α1MG (median value, 5.7 mg/gCr). CONCLUSION: The present data demonstrate that the urine 5MedC level is associated with a reduced renal function and can serve as a novel and potent biomarker for predicting the renal outcome in CKD patients. Further studies will be necessary to elucidate the role of urine DNA methylation in the progression of CKD.

Silver Ions Detection via Nucleolipids Self-Assembly.

A novel hybrid bioinspired amphiphile featuring a cytosine moiety, which self-assembles into liposomes can be used to detect silver ions in aqueous media. The coordination of Ag+ ions by the nucleotide moiety increases membrane rigidity, which enhances the fluorescence of a common reporter, Thioflavin T. Ag+ can be sensed even at trace concentrations (3 ppb) with great specificity over other metals ions. These nucleotide based supramolecular structures can be used to detect silver ions in drinking water, demonstrating the robustness of this approach.

Sagittal plane center of mass movement strategy and joint kinetics during sit-to-walk in elderly fallers.

BACKGROUND: Biomechanical studies have shown that the horizontal center of mass momentum at seat-off during sit-to-walk is reduced among elderly adults. However, the underlying mechanism of this phenomenon is still lacking. The purpose of this study was to examine differences in center of mass movement strategies and lower extremity joint kinetics in the elderly during sit-to-walk. METHODS: Fifteen healthy young adults, fifteen healthy elderly adults (70 years or older) and fifteen elderly fallers performed Timed Up and Go test. Biomechanical data collected from sit-to-walk phase of Timed Up and Go test were analyzed. Outcome measures included center of mass-ankle inclination angles, hip, knee and ankle joint moments, and ground reaction forces of the stance limb. FINDINGS: Results reported here are from 10 participants in each group due to missing force place data. Elderly fallers adopted a movement strategy that included a posterior foot placement at seat-off, a longer duration, and a shorter initial step length. When compared to healthy individuals, elderly fallers demonstrated a greater ankle plantarflexor moment at seat-off. INTERPRETATION: Increased ankle plantarflexor moment in elderly fallers could be a movement strategy to improve stability during sit-to-walk and may be related to the reduced ankle dorsiflexor strength commonly reported in elderly fallers. Results of this study enhance our understanding on the underlying mechanisms of the altered sit-to-walk movement and could aid in developing effective screening and rehabilitation programs to prevent falling in the elderly.

Detection of oxidation products of 5-methyl-2'-deoxycytidine in Arabidopsis DNA.

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2'-deoxycytidine (5-FodC), 5-carboxyl-2'-deoxycytidine (5-CadC), 5-hydroxymethyl-2'-deoxyuridine (5-HmdU), and the (5'S) diastereomer of 8,5'-cyclo-2'-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 10(6) nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.

Methylation stabilizes the imino tautomer of dAMP and amino tautomer of dCMP in solution.

Alkylating agents cause methylation of adenosine and cytidine in DNA to generate 1-methyladenosine and 3-methylcytidine. These modified nucleosides can serve as regulators of cells or can act as agents of mutagenesis depending on the context and the partner enzymes. Solution structures and the chemical interactions with enzymes that lead to their recognition are of inherent interest. At physiological pH, 1-methyladenosine and 3-methylcytidine are presumed to be in the protonated amino forms in the literature. We report the structures, ionization states, and UV resonance Raman spectra of both substrates over a range of pH (2.5-11.0). The Raman excitation wavelength was tuned to selectively enhance Raman scattering from the nucleobase (260 nm) and further specifically from the imino form (210 nm) of 1-me-dAMP. We find that contrary to the general assumption, 1-me-dAMP is present in its neutral imino form at physiological pH and 3-me-dCMP is in the amino form.

Photo-cross-linking of XPC-Rad23B to cisplatin-damaged DNA reveals contacts with both strands of the DNA duplex and spans the DNA adduct.

Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The xeroderma pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photo-cross-linking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photo-cross-linking with denaturation and immunoprecipitation of protein-DNA complexes, we identified the XPC subunit in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B-DNA interaction was largely indirect via its interaction with XPC. Using site specific cross-linking, we determined that the XPC-Rad23B complex is preferentially cross-linked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine 5'-monophosphate) analogue is located to the 5' side of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located to the 3' side of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model in which XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential cross-linking 5' of the cisplatin-damaged site suggests that the XPC-Rad23B complex displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.

Delta-di-carboxybutyl phosphoramidate of 2'-deoxycytidine-5'-monophosphate as substrate for DNA polymerization by HIV-1 reverse transcriptase.

The replacement of the pyrophosphate moiety of 2'-deoxynucleoside triphosphates by non natural delta-dicarboxylic butyl amino acid allows incorporation of natural 2'-deoxycytidine into DNA using HIV-1 reverse transcriptase (RT) as enzyme. In contrast, the 3'-deoxycytidine analogue was not a substrate of the HIV-1 RT.

[Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues].

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique.

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.

Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease.

McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates. We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system. We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein. Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified lambda and T4gt, respectively) and for induction of the E. coli SOS response in the presence of M.HpaII methylation, indicative of DNA damage. Our findings suggest the presence of an N-terminal DNA-binding domain and a C-terminal catalytic nuclease domain connected by a linker region largely tolerant of amino acid insertions. DNA damage inflicted by a functional C-terminal domain is required for restriction of phage T4gt. Disruption of the N-terminal domain abolishes restriction of both substrates. Surprisingly, truncation mutations that spare the N-terminal domain do not mediate DNA damage, as measured by SOS induction, but nevertheless partially restrict M.HpaII-modified lambda in vivo. We suggest a common explanation for this "restriction without damage" and a similar observation seen in vivo with McrB, a component of another of the modified-DNA restriction functions. Briefly, we propose that unproductive site-specific binding of the protein to a vulnerable position in the lambda genome disrupts the phage development program at an early stage. We also identified a single mutant, carrying an insertion in the N-terminal domain, which could fully restrict lambda but did not restrict T4gt at all. This mutant may have a selective impairment in substrate recognition, distinguishing methylated from hydroxymethylated substrates. The study shows that the technically easy insertion scanning method can provide a rich source of functional information when coupled with effective phenotype tests.

Biochemical and mechanistic basis for the activity of nucleoside analogue inhibitors of HIV reverse transcriptase.

HIV encodes an RNA directed DNA polymerase (reverse transcriptase, RT) that is an essential enzyme in the viral replication cycle. This enzyme catalyzes the synthesis of double stranded proviral DNA from single stranded genomic RNA via a bireactant-biproduct mechanism. The functional enzyme purified from virus particles is a complex consisting of two polypeptides of molecular weight 66,000 and 51,000. Two of the four classes of currently approved anti-HIV drugs, the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleoside reverse transcriptase inhibitors (NNRTIs), act by inhibiting this enzyme. In this review each step of DNA synthesis catalyzed by the RT is described and the mechanism of inhibition of catalysis and termination of DNA synthesis by NRTIs is detailed. The individual steps in the catalytic cycle and the effects that the NRTIs have on them have been examined using transient kinetic analysis. The impact of stereoisomerism and resistance mutations on the rate of NRTI triphosphate incorporation (k(pol)), binding in the catalytic complex (K(d)) and the overall efficiency of incorporation (k(pol)/K(d)) are summarized for lamivudine, coviracil and zalcitabine. The results provide insight into the molecular forces and structural features that make these molecules effective inhibitors.

Determination of the DNA methylation level in tumor cells by capillary electrophoresis and laser-induced fluorescence detection.

An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.

Probing the mechanistic consequences of 5-fluorine substitution on cytidine nucleotide analogue incorporation by HIV-1 reverse transcriptase.

Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2'-fluorine substitution on the pre-steady state incorporation of 2'-deoxycytidine-5'-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-beta-L-2',3'-dideoxy-3'-thia-5-fluorocytidine (L-FTC, Emtriva), and (+)-beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC, Reverset), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2' position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.

Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase.

Deoxycytidylate deaminase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of thymidine nucleotides. It also may be involved in the action, as well as the metabolism of anticancer agents. Recently, several L- and D-configuration pyrimidine deoxynucleoside analogs were found to be potent antiviral and antitumor agents. Their interaction with dCMP deaminase as a monophosphate or a triphosphate metabolite is not clear. These include D-nucleoside analogs such as beta-D-2',3'-dideoxycytidine (ddC), beta-2'-fluoro-5-methyl-arabinofuranosyluracil (FMAU), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) as well as L-nucleoside analogs such as beta-L-dioxolane-cytidine (L-OddC), beta-L-2',3'-dideoxy-3'-thiacytidine, beta-L-2',3'-dideoxy-5'-fluoro-3'-thia-cytidine (L-FSddC), beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine, and L-FMAU. None of the L-deoxycytidine analog monophosphates act as substrates or inhibitors. Among these pyrimidine deoxynucleoside analog monophosphates, D-FMAU monophosphate (MP) is the most potent competitive inhibitor, whereas L-FMAUMP has no inhibitory activity. Interestingly, AZTMP and D4TMP also have potent inhibitory activities on dCMP deaminase. Among the dCTP and TTP analogs examined, D- and L-FMAUTP were the most potent inhibitors and had the same extent of inhibitory effect. These results suggest that a chiral specificity for the substrate-binding site may exist, but there is no chiral specificity for the regulator-binding site. This is also supported by the observation that L-OddC and L-FSddC have inhibitory activities as triphosphates but not as monophosphates. None of the D- and L-dCTP analogs activated dCMP deaminase as dCTP. The biological activities of AZT and D4T could be partially attributable to their inhibitory activity against dCMP deaminase by their phosphorylated metabolites, whereas that of ddC and the L-deoxycytidine analogs may not involve dCMP deaminase directly.

5-Methyldeoxycytidine monophosphate deaminase and 5-methylcytidyl-DNA deaminase activities are present in human mature sperm cells.

Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation product 5-mdCMP into dTMP which is further cleaved into thymine and the abasic sugar-phosphate. Both 5-methylcytidine 5' and 3' monophosphates are good substrates for the deaminase. 5-methylcytidine is not a good deaminase substrate and 5-methylcytosine (5mC) is not a substrate. A purified fraction of the deaminase free of nucleases deaminates 5mC present in intact methylated double-stranded DNA. 5-mdCMP deaminase co-purifies on SDS-PAGE with dCMP deaminase and has an apparent molecular weight of 25 kDa. The enzyme requires no divalent cations and has a Km of 1.4 x 10(-7) M for 5-mdCMP and a Vmax of 7 x 10(-11) mol/h/microg protein. The possible biological implications of the deaminase's activities in the present system are discussed.