Search PubMed⌕ Search

Biomedical subjects

J M Essigmann

Publications and source records attributed to J M Essigmann.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.

Base Sequence↗
PubMed1994

Effects of DNA adduct structure and distribution on the mutagenicity and genotoxicity of two platinum anticancer drugs.

cis-Diamminedichloroplatinum(II) (cis-DDP) and cis,trans,cis-ammine(cyclohexylamine)-dibutyratodichloroplatinu m(IV) (ACDDP) are anticancer drugs that bind to DNA, forming replication blocking adducts. ACDDP, probably manifests its cytotoxicity through the metabolite cis-ammine(cyclohexylamine)dichloroplatinum(II) (ACDP). The biological effects of ACDP and cis-DDP were compared by studying polymerase inhibition in vitro and mutagenesis and genotoxicity in vivo in the duplex genome of bacteriophage M13mp18 replicated in Escherichia coli. cis-DDP and ACDP adducts were equally genotoxic within the statistical error limits of the analysis. Survival of genomes platinated by either drug, increased by threefold in cells pretreated with u.v. irradiation to induce the SOS functions of the host. In the M13mp18 lacZ' gene fragment, the mutagenicity of ACDP was lower than that of cis-DDP; the difference was approximately twofold at a dose of two adducts per 370 base-pair mutational target. Mutagenesis by both compounds was SOS-dependent. The structural basis for lower mutagenicity of ACDP is proposed to be its reduced reactivity at d(ApG) sites. This effect is attributed to an orientational isomerism that precludes the formation of one of two possible DNA adducts at d(ApG) residues. The types of mutations induced for both drugs were similar, but they occurred with different distributions. Both compounds induced primarily G-->T transversions at d(GpG) sites whereas G-->A transitions and A-->T transversions, many at d(ApG), d(GpNpG), and d(GpG) sites, were also well represented. The mapping of DNA adducts by DNA synthesis inhibition revealed excellent correlation between the location of DNA lesions and the sites of mutations. Analysis of the distribution of mutations and the distribution of potential platination sites revealed no sequence-dependent mutation hotspots; i.e. mutagenesis was random throughout the lacZ' region of the M13mp18 bacteriophage genome. These results offer insights into the molecular mechanism of mutagenicity of platinum anticancer drugs.

Antineoplastic Agents↗
PubMed1993

Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4-ethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole.

The mutagenic and genotoxic properties of 1,N6-ethenoadenine (epsilon Ade), 3,N4-ethenocytosine (epsilon Cyt), and 4-amino-5-(imidazol-2-yl)imidazole (beta) were investigated in vivo. The former two modified bases are known DNA adducts formed by the human carcinogen vinyl chloride; beta is formed by pyrimidine ring-opening of epsilon Ade. Chemically synthesized deoxyhexanucleotides containing epsilon Ade and beta, d[GCT-(epsilon A)GC], and d[GCT(beta)GC], respectively, were described previously [Biochemistry (1987) 26, 5626-5635]. epsilon Cyt was inserted into an oligonucleotide, d[GCTAG(epsilon C)], by a mild enzymatic synthetic procedure, which avoided exposure of the base to alkaline conditions. 3,N4-Etheno-2'-deoxycytidine 3',5'-bisphosphate coupled with reasonable efficiency (30-40%) to the 3'-nucleoside of an acceptor pentamer, d(GCTAG), in a reaction catalyzed by T4 RNA ligase in the presence of ATP. Each of the three modified hexanucleotides and an unmodified control were inserted into a six-base gap positioned at a known site in the genome of bacteriophage M13-NheI. A nick was placed in the DNA strand opposite that containing the single DNA lesions, enabling the formation of singly adducted single-stranded genomes by denaturation. After transfection of the adducted phage DNAs into Escherichia coli, each of the adducts was found to be genotoxic. The most toxic lesion was beta, which reduced survival of the genome by 97%. epsilon Cyt and epsilon Ade reduced survival by 90% and 65%, respectively. An examination of the surviving phage populations revealed that each of the three adducts was mutagenic. The least mutagenic lesion was epsilon Ade (0.1% of the survivors were mutant), which showed primarily A-->G transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1993

Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diamminedichloroplatinum(II).

The mutagenicity and genotoxicity of cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] (G*G*), the major DNA adduct of the antitumor drug cisplatin, has been investigated in Escherichia coli. A duplex bacteriophage M13 genome was constructed to contain the G*G* adduct at a specific site in the (-) strand. The singly platinated duplex genome exhibited a survival of 22% relative to that of the unplatinated control genomes, and this value rose to 38% in cells treated with ultraviolet light to induce the SOS response. Singly platinated single-stranded genomes were also produced. Replication of the single- and double-stranded genomes in vivo yielded SOS-dependent, targeted mutations at frequencies of 1.3% and 0.16%, respectively. The mutagenic specificity of G*G* in both single- and double-stranded DNA was striking in that 80-90% of the mutations occurred at the 5'-platinated G. Approximately 80% of the mutations were G-->T transversions at that site. A model of mutagenesis is presented to explain this mutational specificity with respect to current understanding of platinum-DNA adduct structure.

Bacteriophage M13↗
PubMed1993

The relationship between the chemical structures and mutagenic specificities of the DNA lesions formed by chemical and physical mutagens.

The mutagenic activities of radiation, alkylating agents, and aromatic amines and amides are examined from the perspective of relating the observed patterns of mutagenesis with the structures of the premutagenic DNA lesions. The general approach taken to establish such relationships has involved the construction of viral or plasmid genomes containing specific DNA adducts. The in vivo replication of such site-specifically modified genomes results in the induction of mutations, which are characterized by DNA sequencing. It is found that the mutation types that arise in these simple systems often match those occurring in tumors produced in mammals exposed intentionally or unintentionally to carcinogenic agents.

Animals↗
PubMed1993

Detection of oxidative damage by 32P-postlabelling: 8-hydroxydeoxyguanosine as a marker of exposure.

Human exposure to reactive oxygen species is unavoidable and has been implicated in the etiology of a number of human diseases. This exposure results in the formation of various modified DNA bases: the promutagenic lesion 8-hydroxydeoxyguanosine (8OHdG), in particular, is a major product. We have developed an assay using ion-pair HPLC and 32P-postlabelling to quantify 8OHdG in human DNA with high specificity and sensitivity. An internal standard is used to account for variations in labelling efficiency. Chemically synthesized 8OHdG 3'-monophosphate and 5'-monophosphate standards were used to optimize the HPLC-32P-postlabelling and TLC separative steps, respectively. The assay was validated using known ratios of 8OHdG to normal nucleotides. The limit of detection is in the range of one 8OHdG residue per 10(6)-10(7) dG residues. Using this procedure, 8OHdG levels of 16-35 8OHdG adducts per 10(5) dG residues have been found in leukocytes isolated from patients who received 180-200 cGy of ionizing radiation. These levels were 2-4-fold greater than those found in an unexposed individual. Since 8OHdG may be formed during DNA extraction and digestion, current procedures for measuring background levels are discussed.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.

Adenine↗
PubMed1992

An intrastrand d(GpG) platinum crosslink in duplex M13 DNA is refractory to repair by human cell extracts.

We have examined the ability of human cell extracts to repair the most frequent DNA adduct caused by the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II). A circular DNA duplex with an intrastrand d(GpG) crosslink positioned at a specific site was synthesized. Human cell extracts were unable to induce repair synthesis in a 29-base-pair region encompassing the adduct or in adjacent regions. The same extracts could repair a single defined 2-acetylaminofluorene lesion in a similar location. When molecules containing the platinum adduct were cleaved by Escherichia coli UvrABC enzyme, human cell extracts could perform repair synthesis at the damaged site, suggesting that human enzymes fail to make incisions near the d(GpG) crosslink but can complete repair once incisions are made. This result indicates that most repair synthesis in DNA damaged with multiple cis-diamminedichloroplatinum(II) adducts takes place at lesions other than the predominant d(GpG) crosslink. These data support the idea that the clinical effectiveness of cis-diamminedichloroplatinum(II) may be explained by the inefficient repair of the major DNA adduct caused by this drug.

Acetoxyacetylaminofluorene↗
PubMed1992

An ultraviolet light-damaged DNA recognition protein absent in xeroderma pigmentosum group E cells binds selectively to pyrimidine (6-4) pyrimidone photoproducts.

The binding specificity was defined of a human ultraviolet light-damaged DNA recognition protein (UV-DRP), the activity of which is absent in some xeroderma pigmentosum complementation group E cells. Our results suggest that cyclobutane pyrimidine dimers (CPDs) are not high affinity UV-DRP binding sites--a finding consistent with other reports on this protein (Hirschfeld et al., (1990) Mol. Cell Biol., 10, 2041-2048). A major role for 6-4 photoproducts in UV-DRP binding was suggested in studies showing that irradiated oligonucleotides containing a T4C UV box sequence, which efficiently forms a TC 6-4 photoproduct, was a superior substrate for the UV-DRP when compared to a similar irradiated oligonucleotide having a T5 sequence. The latter sequence forms CPDs at a much higher frequency than 6-4 photoproducts. In a more direct approach, T4C-containing oligonucleotides complexed with the UV-DRP were separated from the unbound oligonucleotide fraction and the frequencies of 6-4 photoproducts in the two DNA populations were compared. The UV-DRP-bound fraction was highly enriched for the 6-4 lesion over the unbound fraction supporting the conclusion that 6-4 photoproducts are the principal binding cues for the UV-DRP.

Base Sequence↗
PubMed1992

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.

Amino Acid Sequence↗
PubMed1991

Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli.

The qualitative and quantitative features of mutagenesis by two DNA adducts of carcinogenic alkylating agents, O6-methylguanine (m6G) and O4-methylthymine (m4T), were examined in vivo. The deoxyhexanucleotides 5'-GCTAGC-3' and 5'-GCTAGC-3' were synthesized, where the underlined bases are the positions of m4T or m6G, respectively. By use of recombinant DNA techniques, the respective hexanucleotides or an unmodified control were inserted into a six-base gap in the otherwise duplex genome of the Escherichia coli virus M13mp19-NheI. The duplex adducted genome was converted to single-stranded form and introduced into an E. coli strain that was phenotypically normal with regard to m6G/m4T repair, a strain deficient in repair by virtue of an insertion in the gene encoding the Ada-m6G/m4T DNA methyltransferase, or the same two cell lines after challenge with N-methyl-N'-nitro-N-nitrosoguanidine. Treatment with this alkylating agent chemically compromises alkyl-DNA repair functions. The mutation efficiency of m6G was low or undetectable (0-1.7%) in all cell systems tested, owing, we believe, to rapid repair. In striking contrast, the mutagenicity of m4T was high (12%) in cells fully competent to repair alkylation damage and was roughly doubled when those cells were pretreated with N-methyl-N'-nitro-N-nitrosoguanidine to suppress repair. Taken together, these data suggest that m4T is potentially more mutagenic than m6G and, if formed by a DNA methylating agent, may pose a significant threat to the genetic integrity of an organism.

Bacterial Proteins↗
PubMed1991

Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases.

The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.

Base Sequence↗
PubMed1991

3-Methyladenine mutagenesis under conditions of SOS induction in Escherichia coli.

Under conditions of the induced error-prone SOS response in Escherichia coli, N-methyl-N-nitrosourea (MNU) was found to produce a majority of mutations at A.T base pairs. These mutations included mainly A.T----G.C transitions, followed by A.T----T.A transversions and (-1)A.T frameshifts. The possibility that 3-methyladenine (3-MeAde) significantly contributed to these mutations was investigated. MNU mutagenesis under SOS conditions was studied in E. coli strains deficient in 3-MeAde-DNA glycosylase I (TagI), which is the major constitutively expressed repair enzyme for 3-MeAde. In SOS uninduced cells, the lack of 3-MeAde repair did not increase mutagenesis, suggesting that 3-MeAde does not contribute to mutagenesis under these conditions. In SOS-induced cells, by contrast, MNU induced a 5-fold higher mutation frequency in the TagI-deficient cell strains, suggesting that 3-MeAde is indeed an SOS-dependent premutagenic lesion. Although 3-MeAde is mutagenic under SOS conditions, it is possible that its fast repair in repair-proficient cells might result in the loss of the lesion before its mutagenic properties could be realized. Hence, the contribution of 3-MeAde to SOS-dependent mutagenesis in fully repair-proficient cells was also investigated. 3-MeAde lesions were removed from MNU-treated DNA by the purified TagI protein. This prior removal of 3-MeAde had little effect on mutagenesis in SOS-induced or SOS-uninduced cells. Thus, in repair-proficient cells, 3-MeAde is efficiently removed from DNA and does not contribute in a major way to mutagenesis. These results indicate that some other A or T adduct(s) are responsible for the bulk of the mutagenesis observed under SOS-induced conditions.

Adenine↗
PubMed1990

Effect of the antitumor drug cis-diamminedichloroplatinum(II) and related platinum complexes on eukaryotic DNA replication.

An SV40-based in vitro replication system has been used to examine the effects of platinum compounds on eukaryotic DNA replication. Plasmid templates containing the SV40 origin of replication were modified with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) or the inactive analogues [Pt(dien)Cl]+ and trans-DDP. The platinated plasmids were used as templates for DNA synthesis by the DNA polymerases present in cytosolic extracts prepared from human cell lines HeLa and 293. Bifunctional adducts formed by cis- and trans-DDP inhibited DNA replication by 95% at a bound drug to nucleotide ratio [(D/N)b] of less than 9 x 10(-4), in contrast to the monofunctional [Pt(dien)Cl]+ analogues, which required a (D/N)b of 3.4 x 10(-3) for 62% inhibition of DNA replication. An average of two platinum adducts per genome was sufficient for inhibition of DNA replication by cisplatin. When trans-DDP-modified, but not cis-DDP-modified, SV40 origin containing plasmids [(D/N)b = 1.7 x 10(-3)] were allowed to incubate in the 293 cytosolic extracts for 1 h prior to addition of T-antigen to initiate replication, DNA synthesis was restored to 30% of control. This result suggested the presence of an activity in the extracts that reactivates trans-DDP-modified DNA templates for replication. This hypothesis was confirmed by an in vitro nucleotide excision repair assay that revealed activity in 293 and HeLa cell extracts selective for trans-DDP-modified plasmid DNAs. Such selective repair of trans-DDP-damaged DNA in human cells would contribute to its lack of antitumor activity.

Cells, Cultured↗
PubMed1990

Mechanistic studies of ionizing radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome.

T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2'-deoxyguanosine 5',3'-bisphosphate. The acceptor was efficiently converted to the reaction product (greater than 95%), and the final product yield was 50%. Following 3'-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG8-OH) and an unmodified control were 5'-phosphorylated by using [gamma -32P]ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The ligation product contained G8-OH at the 3' residue of an in-frame amber codon (5'-TAG-3') (genome position 6276) of the phage lacZ alpha gene. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (approximately 90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5' and 3' termini. The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin.

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗