Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 67
Filter
Add more filters










Publication year range
1.
Int J Radiat Biol ; 81(6): 445-58, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16308915

ABSTRACT

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.


Subject(s)
Breast Neoplasms/radiotherapy , Cellular Senescence/radiation effects , Tumor Suppressor Protein p53/physiology , Apoptosis/radiation effects , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Cycle/radiation effects , Cell Line, Tumor , Chromosome Aberrations , DNA-Binding Proteins/metabolism , Female , Humans , RNA/analysis , Telomerase/analysis , Telomerase/metabolism , Telomere
2.
J Biol Chem ; 276(26): 24323-30, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11309379

ABSTRACT

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3' --> 5' resection before ligation, was formed. Similar results were seen for a substrate with 5'-overhangs and recessed 3'-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.


Subject(s)
Antigens, Nuclear , DNA Helicases , DNA Repair , DNA-Binding Proteins/physiology , Glycolates/metabolism , Nuclear Proteins/physiology , Androstadienes/pharmacology , Animals , CHO Cells , Cell Extracts/analysis , Cricetinae , DNA-Activated Protein Kinase , DNA-Binding Proteins/genetics , Enzyme Inhibitors/pharmacology , Humans , Ku Autoantigen , Mutation , Nuclear Proteins/genetics , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Oocytes/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Species Specificity , Wortmannin , Xenopus
3.
Anal Biochem ; 289(2): 274-80, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11161322

ABSTRACT

Single- and double-strand breaks bearing 3'-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3'-phosphoglycolate substrates for repair studies, 5'-(32)P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3'-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies.


Subject(s)
Bleomycin/pharmacology , DNA/drug effects , Glycolates/metabolism , Alkaline Phosphatase/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Chromatography, High Pressure Liquid , DNA Damage/drug effects , DNA Repair , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Oligonucleotides/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform Infrared , Time Factors
4.
Cancer Res ; 59(15): 3682-8, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10446982

ABSTRACT

Previous work showed that the DNA double-strand cleaving agents bleomycin and neocarzinostatin were more mutagenic in plateau-phase than in log-phase cells. To determine whether topoisomerase II poisons that produce double-strand breaks by trapping of cleavable complexes would, likewise, induce mutations specific to plateau-phase cells, aprt mutations induced by amsacrine in both log-phase and plateau-phase CHO cells were analyzed. The maximum aprt mutant frequencies obtained were 7 x 10(-6) after treatment with 0.02 microM amsacrine in log phase and 27 x 10(-6) after treatment with 1 microM amsacrine in plateau phase, compared with a spontaneous frequency of < 1 x 10(-6). Base substitutions dominated the spectrum of mutations in log-phase cells, but were much less prevalent in plateau-phase cells. Both spectra also included small deletions, insertions and duplications, as well as few large-scale deletions or rearrangements. About 5% of the log-phase mutants and 16% of the plateau-phase mutants were +1 frameshifts, and all but one of these were targeted to potential free 3' termini of cleavable complexes, as determined by mapping of cleavage sites in DNA treated with topoisomerase II plus amsacrine in vitro. Thus, these insertions may arise from templated extension of the exposed 3' terminus by a DNA polymerase, followed by resealing of the strand, as shown previously for acridine-induced frameshifts in T4 phage.


Subject(s)
Amsacrine/pharmacology , CHO Cells/drug effects , DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Enzyme Inhibitors/pharmacology , Frameshift Mutation , Mutagenesis , Adenine Phosphoribosyltransferase/genetics , Animals , Base Sequence , Cell Division , Cricetinae , DNA/drug effects , Exons/genetics , Genes/drug effects , Molecular Sequence Data , Sequence Deletion , Substrate Specificity , Topoisomerase II Inhibitors
5.
Biochem Pharmacol ; 58(1): 121-31, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10403525

ABSTRACT

Events accompanying sequential exposure of U937 leukemic cells to the deoxycytidine (dCyd) analogs 1-[beta-D-arabinofuranosyl]cytosine (ara-C) or 2',2'-difluorodeoxycytidine (gemcitabine; dFdC) followed by two protein kinase C (PKC) activators [bryostatin 1 (BRY) or phorbol 12'-myristate 13'-acetate (PMA)] exhibiting disparate differentiation-inducing abilities were characterized. A 24-hr exposure to 10 nM BRY or PMA after a 6-hr incubation with 1 microM ara-C or 100 nM dFdC resulted in equivalent increases in apoptosis, caspase-3 activation, and polyADP-ribose polymerase degradation, as well as identical DNA cleavage patterns. BRY and PMA did not modify retention of the lethal ara-C metabolite ara-CTP or alter ara-CTP/dCTP ratios. Unexpectedly, pretreatment of cells with ara-C or dFdC opposed BRY- and PMA-related induction of the cyclin-dependent kinase inhibitors (CDKIs) p21CIP1 and/or p27KIP1. These effects were not mimicked by the DNA polymerase inhibitor aphidicolin or by VP-16, a potent inducer of apoptosis. Inhibition of PKC activator-induced CDKI expression by ara-C and dFdC did not lead to redistribution of proliferating cell nuclear antigen but was accompanied by sub-additive or antagonistic effects on leukemic cell differentiation. Sequential exposure of cells to ara-C followed by BRY or PMA led to substantial reductions in clonogenicity that could not be attributed solely to apoptosis. Finally, pretreatment of cells with ara-C attenuated PMA- and BRY-mediated activation of mitogen-activated protein kinase, an enzyme implicated in CDKI induction. Collectively, these findings suggest that pretreatment of leukemic cells with certain dCyd analogs interferes with CDKI induction by the PKC activators PMA and BRY, and that this action may contribute to modulation of apoptosis and differentiation in cells exposed sequentially to these agents.


Subject(s)
Cell Cycle Proteins , Cyclins/biosynthesis , Cytarabine/pharmacology , Deoxycytidine/analogs & derivatives , Lactones/pharmacology , Leukemia/metabolism , Microtubule-Associated Proteins/biosynthesis , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tumor Suppressor Proteins , Antimetabolites, Antineoplastic/pharmacology , Apoptosis , Bryostatins , Cell Differentiation/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Deoxycytidine/pharmacology , Drug Interactions , Enzyme Activation , Humans , Lactones/antagonists & inhibitors , Leukemia/drug therapy , Leukemia/pathology , Macrolides , Mitogens/pharmacology , Tetradecanoylphorbol Acetate/antagonists & inhibitors , U937 Cells , Gemcitabine
6.
Chem Res Toxicol ; 11(12): 1580-5, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9860504

ABSTRACT

Bleomycin-induced cleavage was examined in several nicked, gapped, or intact duplex DNA substrates, including a structure designed to mimic a proposed singly nicked intermediate in double-strand cleavage. This nicked structure appeared to correctly target the second cleavage event in the complementary strand, resulting in a blunt-ended double-strand break, similar to that induced directly by bleomycin alone in an intact duplex of the same sequence. A one-base-gapped structure was markedly less efficient in correctly targeting bleomycin attack in the complementary strand. The results are consistent with a model of bleomycin-induced double-strand cleavage in which the nick formed by the initial bleomycin attack serves to target secondary attack to a specific position in the complementary strand, resulting in a double-strand break with a defined geometry.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Bleomycin/pharmacology , DNA/drug effects , DNA/chemistry , DNA Fragmentation , Electrophoresis, Polyacrylamide Gel , Guanosine/chemistry , Inosine/chemistry , Oxygen/chemistry
7.
Int J Radiat Biol ; 74(5): 561-4, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9848274

ABSTRACT

Molecular analysis of a bleomycin-induced rearrangement of the aprt gene in CHO cells revealed that it consisted of a nearly perfect three-way exchange among non-homologous sequences, consistent with a mechanism involving cyclically permuted misjoining of the six ends of three double-strand breaks.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Chromosome Aberrations/genetics , DNA/drug effects , Gene Rearrangement , Animals , Antimetabolites, Antineoplastic/toxicity , Base Sequence , Bleomycin/toxicity , CHO Cells/drug effects , Cricetinae , DNA/genetics , Molecular Sequence Data
8.
Environ Mol Mutagen ; 31(4): 333-9, 1998.
Article in English | MEDLINE | ID: mdl-9654242

ABSTRACT

Previous work showed that a CTAAA sequence in the supF gene of the shuttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitrogen mustards melphalan and chlorambucil, and indirect evidence suggested adenine N-3 adducts as premutagenic lesions. In order to characterize the adducts formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTAAA sequence were 3H-labeled. Following treatment of this substrate with [14C]melphalan, thermolabile adducts were depurinated and analyzed by HPLC. Only a single peak bearing both 3H and 14C label was detected and it coeluted with the single major adduct formed by the reaction of melphalan with free adenine base. Various spectrometric analyses of this species were all consistent with its identification as a monofunctional adenine N-3 adduct of melphalan. There was no evidence for any bifunctional adducts involving the labeled adenines. There was little if any release of the adenine N-3 adduct of melphalan by Escherichia coli AlkA protein, under conditions where 3-methyladenine was quantitatively released. The results support the proposal that monofunctional adenine N-3 adducts are intermediates in the generation of A.T-->T.A and A.T-->C.G transversions by aromatic nitrogen mustards.


Subject(s)
Melphalan/metabolism , Melphalan/toxicity , Mutation , NADH, NADPH Oxidoreductases/pharmacology , Base Sequence , DNA Adducts , Escherichia coli/enzymology
9.
Biochemistry ; 37(27): 9827-35, 1998 Jul 07.
Article in English | MEDLINE | ID: mdl-9657696

ABSTRACT

Previous work with Xenopus egg extracts suggested that a wortmannin-sensitive protein phosphorylation event precedes both the removal of modified termini from DNA double-strand break ends and the joining of unmodified ends. To assess the possible role of DNA-dependent protein kinase in effecting this phosphorylation, both DNA end-joining and DNA-stimulated phosphorylation were examined in the presence of various inhibitors. Linear but not supercoiled DNA stimulated the phosphorylation of several endogenous proteins in the extracts, including species of approximately 48, 87, and 96 kDa. This phosphorylation was selectively suppressed by the kinase inhibitors wortmannin, dimethylaminopurine, and LY294002, with a dose response that in each case paralleled the inhibition of DNA end-joining. If wortmannin was added while the end-joining reaction was in progress, end-joining of DNA already present in the reaction continued for some time, but newly added DNA was not joined or processed at all. Ends with 3'-hydroxyl termini were joined much faster than those with 3'-phosphoglycolate termini, although both were equally effective in stimulating protein phosphorylation. The results support a role for DNA-dependent protein kinase in regulating end-joining in vitro, and suggest that at least one of the necessary phosphorylations involves a protein bound at or near the DNA end to be joined. In contrast, nuclear extracts from human cells joined double-strand breaks with normal but not modified termini, and the joining was unaffected by kinase inhibitors, suggesting that the dominant mechanism of end-joining in these extracts did not involve DNA-PK.


Subject(s)
DNA Damage , DNA Repair , DNA-Binding Proteins , DNA/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , DNA-Activated Protein Kinase , Female , Humans , Kinetics , Molecular Sequence Data , Nuclear Proteins , Ovum/enzymology , Phosphorylation , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Tumor Cells, Cultured , Xenopus laevis
10.
Cancer Res ; 57(21): 4699-702, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9354424

ABSTRACT

Among the aprt mutations induced in confluence-arrested Chinese hamster ovary D422 cells by the topoisomerase II poison amsacrine, there was a reciprocal exchange between the aprt gene and an unrelated sequence, accompanied by a chromosomal translocation at the aprt locus. The breakpoints in both parental sequences were hot spots for amsacrine-stimulated DNA cleavage in vitro, and the novel junctions formed were precisely as expected for a mechanism involving reciprocal exchange of topoisomerase II subunits followed by resealing of the breaks and correction of mismatches in the cohesive ends. The results are consistent with a role for direct subunit exchange in the production of chromosomal translocations by topoisomerase poisons, although more complex models involving double-strand breakage and repair could produce reciprocal exchanges of similar specificity.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , DNA Topoisomerases, Type II/genetics , Gene Rearrangement/genetics , Sequence Deletion , Translocation, Genetic , Amsacrine/pharmacology , Animals , Antineoplastic Agents/pharmacology , Base Sequence , CHO Cells/drug effects , Chromosome Mapping , Cricetinae , Molecular Sequence Data
11.
Proc Natl Acad Sci U S A ; 94(22): 12018-23, 1997 Oct 28.
Article in English | MEDLINE | ID: mdl-9342355

ABSTRACT

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually

Subject(s)
Adenine Phosphoribosyltransferase/genetics , DNA Damage , Recombination, Genetic , Translocation, Genetic , Animals , Base Sequence , Bleomycin/pharmacology , CHO Cells , Cricetinae , Free Radicals , Gene Rearrangement , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Mutagenesis , Mutagens/pharmacology , Sequence Analysis, DNA
12.
Radiat Res ; 148(1): 22-8, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9216614

ABSTRACT

Two of the enzymes involved in the response of mammalian cells to ionizing radiation are the DNA-dependent protein kinase and poly(ADP-ribose) polymerase. These enzymes are known to be activated by binding to DNA strand breaks, but previous studies designed to look at strand break specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase, we compared enzyme activation by a series of DNA substrates. Irradiated plasmid DNA activated DNA-dependent protein kinase in a dose-dependent manner. When calculated in terms of the molar concentration of double-strand breaks, the enzyme activation by irradiated DNA was comparable to that by restriction enzyme-cleaved DNA. Linear DNA purified after plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comparison of the enzyme activation by plasmid molecules with different 3'- and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence the response of the DNA-dependent protein kinase. Similar experiments with poly(ADP-ribose) polymerase demonstrated that single- and double-strand breaks activate this enzyme with almost equal efficiency, but because of their greater number, single-strand breaks dominate the response of poly(ADP-ribose) polymerase to irradiated DNA.


Subject(s)
DNA Damage/radiation effects , DNA Fragmentation , DNA Repair , DNA, Bacterial/radiation effects , DNA-Binding Proteins , Poly(ADP-ribose) Polymerases/metabolism , Protein Serine-Threonine Kinases/metabolism , DNA, Single-Stranded , DNA, Superhelical/metabolism , DNA-Activated Protein Kinase , Enzyme Activation , Plasmids , Substrate Specificity
13.
Nucleic Acids Res ; 25(12): 2495-500, 1997 Jun 15.
Article in English | MEDLINE | ID: mdl-9171104

ABSTRACT

In order to assess the possible role of human apurinic/apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3'-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5'-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3'-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3'-phosphoglycolates at a 19 base-recessed 3'-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3'-termini. There was no detectable activity of Ape toward 3'-phosphoglycolates on 1 or 2 base protruding single-stranded 3'-overhangs. The results suggest that both a single-base internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3'-termini, particularly those on 3'-overhangs.


Subject(s)
DNA/metabolism , Lyases/metabolism , Oligodeoxyribonucleotides/metabolism , Phosphoric Diester Hydrolases/metabolism , Base Sequence , DNA Damage , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Glycolates/metabolism , Humans , Kinetics , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Substrate Specificity
14.
Biochemistry ; 36(14): 4248-57, 1997 Apr 08.
Article in English | MEDLINE | ID: mdl-9100020

ABSTRACT

Although the binding of various forms of bleomycin to DNA has been studied extensively, the transient nature of the activated bleomycin species which ultimately attacks DNA has largely precluded direct examination of its physical interactions with DNA. In an attempt to map the minimum binding site required for this species to effect DNA cleavage, several oligonucleotide duplexes were synthesized, each of which contained a single N2-isobutyrylguanine moiety at a specific position in the sequence. These duplexes were end-labeled, and sequence-specific bleomycin-induced cleavage was assessed in each strand of each duplex. Isobutyrylguanine substitution immediately 5' to a primary bleomycin target site suppressed bleomycin-induced cleavage by more than 10-fold. Substitution two bases 5' to a target site suppressed cleavage by about 4-fold, and substitution directly opposite the target site suppressed cleavage by 7-fold. Substitution immediately 3' to the target site, or at other more distant positions 3' or 5', had little or no effect. In cases where cleavage at a primary site was strongly suppressed, cleavage at the corresponding secondary site (the putative site of the second break in a bleomycin-induced double-strand break) was also inhibited, even when the secondary site was several bases away from the isobutyrylguanine substitution. The results suggest that the binding site required for bleomycin-induced DNA cleavage spans a region of approximately 2 or 3 bp in the minor groove, including the base associated with the sugar attacked and one or two bases to its 5' side. Computer-based molecular modeling indicated that these results are consistent with the predictions of recently proposed models in which the bithiazole is intercalated immediately 3' to the cleavage site, and the iron coordination site binds in the minor groove immediately 5' to the cleavage site. Both the empirical data and the modeling studies suggest that N2-isobutyrylguanine substitution effectively blocks the minor groove, but without significantly disturbing DNA secondary structure. Thus, it is proposed that site-specific incorporation of N2-isobutyrylguanine may provide a general method for mapping binding sites of minor groove-binding ligands on DNA.


Subject(s)
Bleomycin/analogs & derivatives , Bleomycin/metabolism , DNA/chemistry , Guanine/analogs & derivatives , Binding Sites , Chromatography, High Pressure Liquid , DNA/metabolism , DNA Damage/genetics , Electrophoresis, Polyacrylamide Gel , Guanine/metabolism , Intercalating Agents , Models, Molecular , Molecular Structure , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Peplomycin/metabolism
15.
Nucleic Acids Res ; 25(6): 1211-8, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9092631

ABSTRACT

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , DNA Damage , Nitrogen Mustard Compounds , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Alkylation , Animals , Base Sequence , CHO Cells , Cricetinae , DNA Methylation , Kinetics , Mechlorethamine/toxicity , Melphalan/toxicity , Models, Molecular , Mutagenesis , Phosphoramide Mustards/toxicity
16.
Mutat Res ; 373(1): 17-29, 1997 Jan 03.
Article in English | MEDLINE | ID: mdl-9015149

ABSTRACT

Treatment of confluence-arrested CHO-D422 cells for 48 h with low concentrations (0.5-3 nM) of the radiomimetic antibiotic neocarzinostatin resulted in an increase in up to 11-fold in the frequency of mutations at the hemizygous APRT locus. Analysis by PCR and DNA sequencing revealed that the mutations were a mixture of base substitutions, small deletions, and large-scale rearrangements. base substitutions occurred preferentially at sequence positions where the drug is known to produce abasic sites with closely opposed strand breaks, e.g., AGT, TGT and AGC, where the abasic site occurs at the underlined base and the strand break occurs opposite the first base in each triplet. These results suggest that the substitutions were produced by replicative bypass of the abasic sites, perhaps during attempted repair of the accompanying strand break. Single-base deletions, which comprised nearly half of all deletions, were targeted to these same sequence positions, suggesting that they may have been generated either by replicative bypass of the abasic sites, or by end-joining repair of double-strand breaks, which are induced the same sites. Quantitative analysis of neocarzinostatin-induced damage to APRT DNA in vitro confirmed the association between lesions involving concommitant damage to both DNA strands, and mutations. The results are consistent the hypothesis that agents which induce such bistranded DNA damage can produce biologically significant levels of mutagenesis even in nondividing cells.


Subject(s)
Adenine Phosphoribosyltransferase/genetics , Antibiotics, Antineoplastic/pharmacology , Mutagenesis/drug effects , Sequence Deletion/drug effects , Zinostatin/pharmacology , Animals , Base Sequence , CHO Cells , Cells, Cultured , Chromosome Mapping , Cricetinae , DNA/drug effects , DNA Damage , DNA Fragmentation/drug effects , DNA Repair , Models, Biological , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis, DNA
17.
Int J Radiat Biol ; 70(6): 623-36, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8980659

ABSTRACT

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what specificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a specific site in an SV40-based shuttle vector, by ligating purified 3'-phosphoglycolate-terminated oligonucleotides into 3' recessed ends generated in the linearized vector. These terminally blocked linear vectors were efficiently repaired and replicated when transfected into simian CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a significant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglycolate removal, followed by blunt-end ligation. For the analogous 3'-hydroxyl terminated break, the fraction of simple blunt-end ligations was considerably higher. For a phosphoglycolate-terminated vector with cohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate removal. For all these substrates, the remaining repair joints showed small or large deletions from one or both of the ends, usually with apparent annealing at short (1-4-base) homologies. The results suggest that while breaks with 3'-phosphoglycolates can be repaired, these blocked termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity. The presence of cohesive ends appears to improve markedly the fidelity of rejoining for terminally blocked double-strand breaks.


Subject(s)
DNA Repair/genetics , DNA/genetics , Genetic Vectors/genetics , Glycolates/chemistry , Animals , Base Sequence , Cells, Cultured , DNA/chemistry , DNA Damage/genetics , DNA Repair/physiology , DNA Restriction Enzymes , Molecular Sequence Data , Polyomavirus/genetics , Transfection
18.
Hum Mol Genet ; 5(10): 1657-61, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8894703

ABSTRACT

Biotinidase is responsible for recycling the vitamin biotin from biocytin that is formed after the proteolytic degradation of the biotin-dependent carboxylases. We have identified a deletion/insertion mutation within exon D of the human biotinidase gene in a child with biotinidase deficiency. The mutation causes a frame shift and premature termination which are predicted to result in a truncated protein. We propose that the mutation occurred during DNA replication by either of two mechanisms. Both mechanisms involve formation of a quasipalindromic hairpin loop in the template and dissociation of DNA polymerase alpha. This mutation supports the formation of palindromic structures as a possible cause of deletions in eukaryotes, and supports the proposal, derived from in vitro studies, that polymerase alpha may preferentially arrest or dissociate at specific template sequences.


Subject(s)
Amidohydrolases/deficiency , Chromosomes, Human, Pair 3 , Chromosomes, Human, Pair 7 , Amidohydrolases/genetics , Base Sequence , Biotinidase , DNA, Complementary/analysis , DNA, Complementary/genetics , Exons/genetics , Gene Deletion , Homozygote , Humans , Infant , Male , Molecular Sequence Data , Nucleic Acid Conformation
19.
Mutat Res ; 355(1-2): 71-89, 1996 Aug 17.
Article in English | MEDLINE | ID: mdl-8781578

ABSTRACT

Bleomycin and the enediyne antibiotics effect concerted, simultaneous site-specific free radical attack on sugar moieties in both strands of DNA, resulting in double-strand breaks of defined geometry and chemical structure, as well as abasic sites with closely opposed strand breaks. The hypersensitivity of several mammalian double-strand break repair-deficient mutants to these agents confirms the role of these double-strand breaks in mediating cytotoxicity. In bacteria, mutagenesis by both bleomycin and neocarzinostatin appears to result from replicative bypass of abasic sites, the repair of which is blocked by the presence of closely opposed strand breaks. However, in mammalian cells, such abasic sites decompose to form double-strand breaks, and mutagenesis consists primarily of small deletions, large deletions, and gene rearrangements, all of which probably result from errors in double-strand break repair by a nonhomologous end-joining mechanism. Studies with the radiomimetic antibiotics emphasize the importance of this end-joining repair pathway, and these agents provide useful probes of its mechanistic details, particularly the effects of chemically modified DNA termini on repair.


Subject(s)
Antibiotics, Antineoplastic/toxicity , Bleomycin/toxicity , DNA Damage , Mutagenesis , Mutagens/toxicity , Zinostatin/toxicity , Animals
20.
J Biol Chem ; 271(33): 19660-3, 1996 Aug 16.
Article in English | MEDLINE | ID: mdl-8702667

ABSTRACT

Both mammalian cells and Xenopus eggs possess activities for the joining of nonhomologous DNA ends, and such activities may play a major role in double-strand break repair. In order to dissect the biochemical processing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3'-phosphoglycolate termini were constructed and treated with Xenopus egg extracts. These vectors were rejoined by the extracts at rates 30-100 times slower than comparable 3'-hydroxyl vectors. Vectors with blunt or cohesive 3'-phosphoglycolate ends yielded single repair products corresponding to simple phosphoglycolate removal followed by ligation, while a vector with mismatched ends was also rejoined but yielded a mixture of products. Addition of the kinase inhibitors wortmannin and dimethylaminopurine not only blocked rejoining, but also suppressed phosphoglycolate removal, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxidatively modified ends are repaired in Xenopus eggs by a highly conservative and stringently regulated end-joining pathway, in which all biochemical processing of the breaks is contingent on both end alignment and a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effecting this phosphorylation.


Subject(s)
Androstadienes/pharmacology , DNA Repair/drug effects , Animals , Base Sequence , DNA/metabolism , DNA Damage , Enzyme Inhibitors/pharmacology , Free Radicals , Molecular Sequence Data , Ovum/enzymology , Phosphatidylinositol 3-Kinases , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Wortmannin , Xenopus laevis
SELECTION OF CITATIONS
SEARCH DETAIL
...