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PubMed2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

UNLABELLED: Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. SIGNIFICANCE STATEMENT: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

Preprint↗

PubMed1998

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D.

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an approximately 465-kD catalytic subunit, DNA-PKcs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PKcs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.

3T3 Cells↗

PubMed1992

Anticellular and antitumor activity of duocarmycins, novel antitumor antibiotics.

The anticellular and antitumor activities of novel antitumor antibiotics, duocarmycins (DUMs), were examined against human and murine tumor cells. DUMs consist of five compounds, A, B1, B2, C1 and C2, which possess a pharmacophore similar to that of CC-1065, a previously isolated antibiotic. Among them, DUMA exhibited ultrapotent growth-inhibitory activity with an IC50 value of 6 pM against human uterine cervix carcinoma HeLa S3 cells. DUMA and DUMB1 also inhibited the growth of adriamycin (ADM)-resistant lines of human nasopharynx carcinoma KB cells and breast carcinoma MCF-7 cells as well as their sensitive lines. DUMs inhibited the growth of s.c.-inoculated murine tumors such as B16 melanoma, sarcoma 180, M5076 sarcoma and colon 26. DUMs were also significantly effective in increasing the lifespan of i.p.-inoculated B16 melanoma-bearing mice, although their effect was marginal against other i.p.-inoculated tumors. As a whole, DUMB1 exhibited superior activity to the other four compounds. DUMB1 rapidly inhibited the incorporation of [3H]-TdR into macromolecules of HeLa S3 cells as compared with that of [3H]UR or [3H]leucine. DNA strand breaks were detected in DUMB1-treated HeLa S3 cells by agarose gel electrophoresis with a contour-clamped homogeneous electric field apparatus. These results indicate that DUMs possess interesting biological activities as DNA-targeting antitumor antibiotics.

Animals↗

PubMed1999

Cross-resistance to ionizing radiation in a murine leukemic cell line resistant to cis-dichlorodiammineplatinum(II): role of Ku autoantigen.

cis-Dichlorodiammineplatinum(II) (CDDP; cisplatin) is commonly used in combination with ionizing radiation (IR) in the treatment of various malignancies. In vitro, many observations suggest that acquisition of CDDP resistance in cell lines confers cross-resistance to IR, but the molecular mechanisms involved have not been well documented yet. We report here the selection and characterization of a murine CDDP-resistant L1210 cell line (L1210/3R) that exhibits cross-resistance to IR because of an increased capacity to repair double-strand breaks compared with parental cells (L1210/P). In resistant cells, electrophoretic mobility shift assays revealed an increased DNA-end binding activity that could be ascribed, by supershifting the retardation complexes with antibodies, to the autoantigen Ku. The heterodimeric Ku protein, composed of 86-kDa (Ku80) and 70-kDa (Ku70) subunits, is the DNA-targeting component of DNA-dependent protein kinase (DNA-PK), which plays a critical role in mammalian DNA double-strand breaks repair. The increased Ku-binding activity in resistant cells was associated with an overexpression affecting specifically the Ku80 subunit. These data strongly suggest that the increase in Ku activity is responsible for the phenotype of cross-resistance to IR. In addition, these observations, along with previous results from DNA-PK- mutant cells, provide evidence in favor of a role of Ku/DNA-PK in resistance to CDDP. These results suggest that Ku activity may be an important molecular target in cancer therapy at the crossroad between cellular responses to CDDP and IR.

Animals↗

PubMed1998

Cloning and biochemical characterization of TAF-172, a human homolog of yeast Mot1.

The TATA binding protein (TBP) is a central component of the eukaryotic transcriptional machinery and is the target of positive and negative transcriptional regulators. Here we describe the cloning and biochemical characterization of an abundant human TBP-associated factor (TAF-172) which is homologous to the yeast Mot1 protein and a member of the larger Snf2/Swi2 family of DNA-targeted ATPases. Like Mot1, TAF-172 binds to the conserved core of TBP and uses the energy of ATP hydrolysis to dissociate TBP from DNA (ADI activity). Interestingly, ATP also causes TAF-172 to dissociate from TBP, which has not been previously observed with Mot1. Unlike Mot1, TAF-172 requires both TBP and DNA for maximal (approximately 100-fold) ATPase activation. TAF-172 inhibits TBP-driven RNA polymerase II and III transcription but does not appear to affect transcription driven by TBP-TAF complexes. As it does with Mot1, TFIIA reverses TAF-172-mediated repression of TBP. Together, these findings suggest that human TAF-172 is the functional homolog of yeast Mot1 and uses the energy of ATP hydrolysis to remove TBP (but apparently not TBP-TAF complexes) from DNA.

Adenosine Triphosphatases↗

PubMed1996

The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight.

The phenomenon of DNA-dependent protein phosphorylation was first described little over 10 years ago. Since then a DNA-dependent protein kinase, DNA-PK, has been purified from human cells and many of its biochemical properties have been characterized. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a DNA-targeting protein, Ku. When assembled on a suitable DNA molecule, the DNA-PK holoenzyme acts as a serine/threonine protein kinase that in vitro phosphorylates many DNA binding and non-binding proteins and transcription factors. Recent genetic studies point strongly to functions in DNA double-strand break repair and V(D)J recombination. In addition, biochemical studies suggest a role in the regulation of transcription. Here we discuss, from a historical perspective, the events leading up to our current understanding of the function of DNA-PK, including recent results from our own studies suggesting the involvement of DNA-PK in apoptosis and in viral infection of human cells.

Animals↗

PubMed1998

CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku.

Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L276-L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136-2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.

Antigens, Nuclear↗

PubMed1996

Novel aromatic urea derivatives with DNA-binding ability.

Several aromatic urea derivatives were designed and synthesized as DNA-targeting agents. N,N'-Dimethyl-N,N'-bis[(4-amidylphenyl)aminocarbonyl]-2,6-di aminopyridine (1) and 1,3-bis[5-(glycylamino)pyrid-2-yl]urea (3) showed remarkable DNA-binding abilities as determined by ultrafiltration assay using calf thymus DNA, their potencies being equal to and half that of netropsin, respectively. Compound 1 inhibited the proliferation of both L1210 cells and KB cells with similar IC50 values to netropsin.

Animals↗

PubMedDate not supplied

Phage T4 DNA [N6-adenine] methyltransferase: kinetic studies using oligonucleotides containing native or modified recognition sites.

The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

Bacteriophage T4↗

PubMed1994

Free-radical repair by a novel perthiol: reversible hydrogen transfer and perthiyl radical formation.

2-(3-Aminopropyl-amino) ethaneperthiol (RSSH, the perthiol analogue of the thiol radioprotector, WR-1065) reacts with the alpha-hydroxy alkyl radical (CH3)2C.OH by donating a hydrogen atom as indicated by the characterization of perthiyl radicals (RSS.; lambda max approximately 374 nm, epsilon 374 approximately 1680 +/- 20 dm3 mol-1 cm-1) by pulse radiolysis. The perthiyl radical abstracts a hydrogen from the alcohol to establish a reversible hydrogen-transfer equilibrium. This equilibrium lies predominantly on the side of radical repair since the rate constants for the forward and reverse reactions at pH 4 are: kappa(RSSH+(CH3)2C.OH) = (2.4 +/- 0.1) x 10(9) dm3 mol-1 s-1 and kappa(RSS.+(CH3)2CHOH) = (3.8 +/- 0.3) x 10(3) dm3 mol-1 s-1 respectively. The pKa (RSSH<-->RSS(-)+H+) = 6.2 +/- 0.1 was determined from the pH dependence of the rate of perthiol repair. Identical experiments have been performed with WR-1065 allowing a direct comparison of free-radical repair reactivity to be made with the parthiol analogue. At pH approximately 7.4 the reactivities of the thiol and perthiol were similar, both repairing the alcohol radical with a rate constant of approximately (2.4 +/- 0.1) x 10(8) dm3 mol-1 s-1. However, at pH 5 whilst the hydrogen-donation rate of the thiol was 15-20% higher than at pH 7.4, the perthiol reactivity was over an order of magnitude higher. The thermodynamic driving force for the observed enhanced free-radical repair reactivity of RSSH compared to RSH is attributed to the resonance stabilization energy of 8.8 kJ mol-1 within the RSS. radical. These results indicate a possible application of RSSH/RSS- as DNA-targeted antioxidants or chemoprotectors.

Free Radicals↗

PubMed1998

Formation of a major DNA adduct of the mitomycin metabolite 2,7-diaminomitosene in EMT6 mouse mammary tumor cells treated with mitomycin C.

Treatment of EMT6 mouse mammary tumor cells with [3H]mitomycin C (MC) results in the formation of six major DNA adducts, as described earlier using an HPLC assay of 3H-labeled products of enzymatic hydrolysis of DNA isolated from MC-treated cells. Four of these adducts were identified as monofunctional and bifunctional guanine-N2 adducts in the minor groove of DNA. In order to establish relationships between individual types of MC-DNA adducts and biological responses it is necessary to identify all of the adducts formed in cells. To this end we have now identified a predominant, previously unknown adduct formed in MC-treated EMT6 cells as a derivative not of MC, but of 2,7-diaminomitosene (2,7-DAM), the major bioreductive metabolite of MC. Rigorous proof demonstrates that it is a DNA major groove, guanine-N7 adduct of 2,7-DAM, linked at C-10 to DNA. The adduct is relatively stable at ambient temperature, but is readily depurinated upon heating. Its isolation from MC-treated cells indicates that MC is reductively metabolized to 2,7-DAM, which then undergoes further reductive activation to alkylate DNA, along with the parent MC. Low MC:DNA ratios were identified as a critical factor promoting 2,7-DAM adduct formation in an in vitro model calf thymus DNA/ MC/reductase model system, as well as in MC-treated EMT6 cells. The 2,7-DAM-guanine-N7 DNA adduct appears to be relatively noncytotoxic, as indicated by the dramatically lower cytotoxicity of 2,7-DAM in comparison with MC in EMT6 cells. Like MC, 2,7-DAM exhibited slightly greater cytotoxicity to cells treated under hypoxic as compared to aerobic conditions. However, 2,7-DAM was markedly less cytotoxic than MC under both aerobic and hypoxic conditions. Thus, metabolic reduction of MC to 2,7-DAM represents a detoxification process. The differential effects of MC-DNA and 2,7-DAM-DNA adducts support the concept that specific structural features of the DNA damage may play a critical role in the cytotoxic response to a DNA-targeted chemotherapeutic agent.

Animals↗

PubMed1992

5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5-nitraquine), a new DNA-affinic hypoxic cell radiosensitizer and bioreductive agent: comparison with nitracrine.

Targeting of electron-affinic radiosensitizers to DNA via noncovalent binding (e.g., intercalation) may offer the potential for increasing sensitizing efficiency. However, it has been suggested that high-affinity DNA binding may compromise sensitization by restricting the mobility of sensitizers along the DNA, and by decreasing rates of extravascular diffusion in tumors. The weak DNA intercalator nitracrine (1-NC) is a more efficient radiosensitizer than related nitroacridines with higher DNA-binding affinities (Roberts et al., Radiat. Res. 123, 153-164, 1990). The present study investigates whether electron-affinic agents of even lower DNA-binding affinity may be superior to nitroacridines. The quinoline analog of 1-NC, 5-nitraquine (5-NO), was shown to have an intrinsic association constant for calf thymus DNA in 20 mM phosphate buffer which was 12-fold lower than that of 1-NC. 5-Nitraquine was not accumulated as efficiently as 1-NC by AA8 cells, but, despite a similar one-electron reduction potential, was 2- to 3-fold more potent than 1-NC as a hypoxia-selective radiosensitizer in vitro when compared on the basis of average intracellular concentration. Thus the radiosensitizing potency of 5-NQ appears not to be compromised by its low DNA-binding affinity. The cytotoxic mechanisms of 5-NQ and 1-NC appear to be similar (hypoxia-selective formation of DNA monoadducts), but 5-NQ is 1200-fold less potent than 1-NC as a cytotoxin. Despite this advantage, 5-NQ was not active in vivo as a radiosensitizer in SCCVII tumors. This lack of activity appears to be due to its relatively high toxicity in vivo (intraperitoneal LD50 of 105 mumol kg-1 in C3H/HeN mice), high one-electron reduction potential (-286 mV), and rapid metabolism to the corresponding amine in mice. The in vitro therapeutic index (hypoxic radiosensitizing potency/aerobic cytotoxic potency) of this weak DNA binder was lower than that of the non-DNA targeted radiosensitizer misonidazole, suggesting that DNA targeting enhances cytotoxicity more than radiosensitization. Development of useful DNA-targeted radiosensitizers may require the exploitation of DNA binding modes different from those of the nitroacridines and nitroquinolines.

Aminoquinolines↗

PubMed1992

[Effective complementarily-addressed photomodification of nucleic acids by oligonucleotide derivatives, containing aromatic azido groups].

Highly effective site-specific photomodification of a DNA-target was carried out with oligonucleotide reagents carrying aromatic azido groups. Oligonucleotide derivatives with a photoactive function R on the 5'-terminal phosphate and at C-5 atom of deoxyuridine were synthesized: R1NH(CH2)3NHpd(TCCACTT) and d(ULNHRCCACTT), where R1 is p-azidotetrafluorobenzoyl, R2 is 2-nitro, 5-azidobenzoyl, R3 is p-azidobenzoyl; LNH = -CH2NH-, -CH2OCH2CH2NH- or -CH2NHCOCH2CH2NH-. The prepared compounds form stable complementary complexes and effect site-specific photomodification of the target DNA. The modification of pentadecanucleotide d(TAAGTGGAGTTTGGC) with the reagents was investigated. Maximum extent of modification strongly depended on the reagent's type, the photoreagent with R1 being the most effective. Whatever the binding site was, this agent provided a 65-70% modification in all cases except LNH = -CH2NH-, when the yield was twice lower. For the reagents bearing R1 the modification sites were identified. Selective modification at the G9 residue was detected in the case of LNH = -CH2OCH2CH2NH- and when a photoactive group was linked to the terminal phosphate.

Autoradiography↗

PubMed1988

[Complementary addressed modification of single- and double-stranded DNA by alkylating derivatives of oligonucleotides isolated by partial DNA fragmentation].

Reagents for complementary addressed modification of nucleic acids are proposed to be synthesized on the base of oligonucleotides obtained by partial chemical fragmentation of DNA. The alkylating 4-(N-2 chlorethyl-N-methylamino) benzyl-5'-phosphamide derivatives of 5'-[32P]-labelled oligonucleotides obtained from single and double-stranded DNA cloned in bacteriophage M13 mp9 have been synthesized. The alkylated derivatives of oligonucleotides selectively modify the complementary tracts of single-stranded DNA-target. They are also able to modify the complementary regions in double-stranded supercoiled plasmid DNA.

Alkylating Agents↗

PubMed1995

[Cleavage of a double-stranded DNA target by bleomycin derivatives of oligonucleotides, forming a ternary complex].

Hexadecathymidylate derivatives, containing covalently-bound antitumor antibiotic bleomycin A5, were shown to form a triple-helix complex with double-strand 30-bp DNA-target and to carry out within this complex complementary-addressed DNA modification. Fivefold excess of reagent in relation to target leads to non-specific cleavage mainly of pyrimidine-rich DNA strand. Total degrees of the target-strand cleavage by 5'- and 3'-bleomycin derivatives of hexadecathymidylate were 25 and 35% for purine-rich strand and 47 and 36% for pyrimidine-rich strand. Degrees of non-specific cleavage by 5'-bleomycin derivative of hexadecanucleotide that does not form triple-helix were 6 and 16% for purine- and pyrimidine-rich strands, respectively. Comparison of these data has shown that site-specific cleavage prevailed nonspecific one. Triplex of 5'-bleomycin derivative with DNA melted by 5 degrees C lower (m.p. 40 degrees C) than the similar triplex of hexadecathymidylate. Temperature lowering from 50 to 20 degrees C increases the DNA-cleavage degree according to the increase in the part of target molecules involved in triple-helix formation.

Base Sequence↗

PubMed1995

Recruitment and proliferation of CD8+ T cells in respiratory virus infections.

The dramatic increase in the cellularity of the mediastinal lymph nodes (MLN) of mice infected intranasally (i.n.) with influenza viruses is a consequence of both recruitment and proliferation. As many as 20% of the CD8+ subset in the MLN can be shown to be in S or G2 + M phase at 6 days after i.n. challenge with the HKx31 influenza A virus, the percentage of of cycling cells being approximately five times greater for the activated/memory substantial evidence of apoptosis was found for CD8+ T cells recovered from the MLN and lung, particularly at 5 and 7 days after infection. Less than 1/100 of the proliferating T cells could be shown, by limiting dilution analysis (LDA), to be influenza virus-specific CD8+ cytotoxic T lymphocyte precursors (CTLp). A single, low dose (20 mg/kg) of the DNA-targeted drug cyclophosphamide (Cy) caused a massive decrease in frequency for the responding CD8+ CTLp, though the mice survived infection with the HKx31 virus and there was no long-term exhaustion of the CTLp pool in the MLN, spleen, or lung. The Cy treatment was also followed by a smaller reduction in the prevalence of memory CTLp (specific for Sendai virus) that were present concurrently in the regional lymph node, indicating that a measure of bystander activation is occurring. The experiments show that respiratory virus infections have no negative impact on established T cell memory, and that there is no phase of transient exhaustion in the acute virus-specific CTLp response in this localized infection.

Animals↗

PubMed1994

Radiosensitizing and cytotoxic properties of DNA targeted phenanthridine-linked nitroheterocycles of varying electron affinities.

2-Nitroimidazoles targeted to DNA via intercalation have previously been shown to be as much as 10-100 times more efficient on a molar basis than the untargeted nitroimidazole, misonidazole, in vitro as hypoxic cell selective radiosensitizers and cytotoxins based on extracellular concentrations. In this work the effect of varying the nitroaromatic group has been examined through the preparation of a DNA-targeted 4-nitroimidazole (4-MeNLP-3), a 5-nitroimidazole (5-NLP-3) and a 5-nitrofuran (FEP-2) linked to phenanthridinium ions. With the previously synthesized 2-nitroimidazoles, this provides a series of DNA targeted compounds of varying electron affinity as well as structure at the nitroaromatic position. The present series of compounds was tested for partition coefficient, DNA binding ability, reduction potentials and in vitro radiosensitizing and cytotoxic abilities. The 4-nitro compound displays good radiosensitizing ability, with an SER of approximately 2, despite its low reduction potential, -551 mV. However, hypoxia selective toxicity is lost at the lower reduction potentials presumably due to redox-independent mechanisms. The results obtained indicate that targeting such compounds to DNA diminishes the dependency of radiosensitizing and cytotoxic properties on reduction potential and may allow significant uncoupling of toxicity from radiosensitizing ability.

Animals↗

PubMed1994

The use of 32P-postlabelling to detect DNA adducts produced by experimental anticancer drugs: DNA-directed nitrogen mustards.

DNA alkylation by four acridine-linked 'DNA-targeted' aniline mustard derivatives has been studied by 32P-postlabelling. P1 nuclease digestion proved much more efficient than butanol extraction for enhancing the yield of adducted bases for these somewhat hydrophilic compounds. The yield of adducts was maximal after approximately 4 h digestion with micrococcal nuclease/spleen phosphodiesterase and remained relatively constant after that up to 24 h, suggesting that the adducts formed are stable under these conditions. There was some variation in the rates of phosphorylation of the adducts by T4 polynucleotide kinase, with optimal labelling generally occurring after 1 h. The (CH2)5O-linked half-mustard derivative 1 gave five nucleotide 3'-diphosphate adduct spots with calf thymus DNA. Two of these were identified as the adenine N1 and N3 adducts, corresponding to those previously identified as the main base adducts formed by 1 following acid digestion studies. The corresponding full mustard also gave five adduct spots. In contrast, the (CH2)3-linked half-mustard 3 gave only two adduct spots, the most intense of which was identified as a guanine adduct. The corresponding full mustard 4 gave three adduct spots, two of which were identified as guanine adducts. These results agree well with those obtained for the same compounds by the more tedious methods of acid digestion to base adducts, followed by isolation on HPLC, and show that the technique of 32P-labelling can be usefully applied to the study of alkylation of DNA by this class of 'targeted' mustards.

Aniline Mustard↗