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S J Lippard

Publications and source records attributed to S J Lippard.

At least 73 records · Page 4Linked to original sources
PubMed1998

Telomere loss in cells treated with cisplatin.

Telomeres play an important role in the immortalization of proliferating cells. The long tandem repeats of 5'-TTAGGG-3' sequences in human telomeres are potential targets for the anticancer drug cisplatin, which forms mainly intrastrand d(GpG) and d(ApG) cross-links on DNA. The present study reveals that telomeres in cisplatin-treated HeLa cells are markedly shortened and degraded. A dose that killed 61% of the cells but allowed one round of cell division resulted in shortened telomeres before the induction of apoptosis. Higher doses of cisplatin halted cell cycle progression during the first S phase and triggered apoptosis followed by degradation of telomere repeats. A model in which both cell division with incomplete replication and induction of apoptosis by cisplatin could occur was devised to explain the drug-induced telomere loss.

Apoptosis↗
PubMed1998

Human testis-determining factor SRY binds to the major DNA adduct of cisplatin and a putative target sequence with comparable affinities.

cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a widely used anticancer drug that is most effective against tumors of the testis. Although cisplatin is believed to mediate its cytotoxicity through the formation of DNA adducts, the precise biochemical mechanisms underlying its antitumor activity and selectivity for testicular tumors remain elusive. Of significance are the high-mobility group (HMG) domain and other proteins that bind specifically to cisplatin-DNA adducts. The present study focuses on the testis-specific HMG domain protein human SRY (hSRY). The full-length hSRY protein and its HMG domain region alone were expressed in Escherichia coli and purified to homogeneity. The affinities and specificities of full-length hSRY and the hSRY-HMG domain for 20 bp DNAs containing a single cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] intrastrand cross-link or a putative hSRY target site in the CD3epsilon gene enhancer (AACAAAG) were determined in electrophoretic mobility shift assays. Full-length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a Kd(app) of 120 +/- 10 nM and exhibited a 20-fold specificity over unmodified DNA. The HMG domain of hSRY was sufficient for this interaction. The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand cross-link with higher affinity [Kd(app) = 4 +/- 0.7 nM] but with lower specificity (5-fold) than the full-length protein. The affinities of full-length hSRY and the hSRY-HMG domain for a single cisplatin-DNA adduct were comparable to those for the putative target sequence AACAAAG. These data suggest that cisplatin-DNA adducts may compete with specific DNA sequences in vivo for the binding of human SRY. A possible role for this testis-specific protein in the cytotoxicity and organotropic specificity of cisplatin for testicular tumors is proposed.

Binding, Competitive↗
PubMed1997

Binding of tsHMG, a mouse testis-specific HMG-domain protein, to cisplatin-DNA adducts.

The anticancer drug cisplatin is particularly effective against testicular tumors. Although the clinical consequences of cisplatin chemotherapy are well-known, the precise mechanism of action remains elusive. Specific recognition of cisplatin-damaged DNA by a class of proteins containing the high-mobility group (HMG) domain DNA-binding motif could play a role in mediating the cytotoxicity of the drug. This study presents a quantitative investigation of binding of the murine testis-specific high-mobility group protein tsHMG to DNA modified by cisplatin. The binding affinity and specificity of this protein to a site-specific 1,2-d(GpG) cisplatin-DNA intrastrand cross-link in a 20 bp probe were determined. A value for the apparent dissociation constant, Kd(app), of 24 +/- 5 nM was obtained by gel mobility shift assays. Binding competition assays with the corresponding unmodified 20 bp probe gave a ratio (rho) of nonspecific to specific Kd(app) values of 230. A polypeptide containing tsHMG domain A (residues 1-82) was expressed and purified to homogeneity. This domain alone was sufficient for specific recognition of cisplatin-modified DNA with a Kd(app) of 300 +/- 50 nM and a rho of 20, a comparatively high discrimination factor. DNase I interference analysis of the adduct-containing strand revealed that tsHMG binding extends over 14 nucleotides, centered around the platinated bases. The domain A polypeptide protection pattern covers a slightly smaller area of 13 nucleotides. The binding affinity and specificity of tsHMG for cisplatin-modified DNA are exceptional compared to those of other HMG-domain proteins studied previously. The possible relevance of these findings to the mechanism of action of cisplatin is discussed.

Animals↗
PubMed1997

DNA sequence context and protein composition modulate HMG-domain protein recognition of cisplatin-modified DNA.

Proteins containing the high mobility group (HMG) DNA-binding domain form specific complexes with cisplatin-modified DNA which shield the major intrastrand d(GpG) and d(ApG) cross-links from excision repair. The molecular basis for the specificity of binding was investigated for the two isolated domains of HMG1 with a series of 15-bp oligonucleotides, d(CCTCTCN1G*G*N2TCTTC). (GAAGAN3CCN4GAGAGG), where asterisks denote N7-modification of guanosine with cisplatin. Alteration of the nucleotides flanking the platinum lesion modulated HMG1domA recognition in this series by over 2 orders of magnitude and revealed an unprecedented preference for N2 = dA > T > dC. The flanking nucleotide preference for HMG1domB interaction with this oligonucleotide series was less pronounced and had only a 20-fold range of binding affinities. For the N1 = N2 = dA 15-bp probe, 100-fold stronger binding occurred with HMG1domA (Kd = 1.6 +/- 0.2 nM) compared to HMG1domB (Kd = 134 +/- 18 nM). The platinum-dependent recognition of the N1 = N2 = dA 15-bp probe saturates at 1 equiv of HMG1domA and is highly specific, as evidenced by the 1000-fold decrease in HMG1domA binding affinity for the corresponding unplatinated oligonucleotide. HMG domains were unable to bind specifically to cisplatin-modified DNA-RNA hybrids, revealing the need for a deoxyribose sugar backbone for specific complex formation with HMG-domain proteins. Protein-DNA contacts which may account for these observed binding preferences are proposed, and potential implications for the biological processing of cisplatin-DNA adducts are discussed.

Amino Acid Sequence↗
PubMed1997

Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 A resolution. The enzyme is composed of two copies each of three subunits (alpha 2 beta 2 gamma 2), and all three subunits are almost completely alpha-helical, with the exception of two beta hairpin structures in the alpha subunit. The active site of each alpha subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4 degrees C, a bridging acetate ion, which is replaced at -160 degrees C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural differences identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position.

Crystallography, X-Ray↗
PubMed1996

Repair of cisplatin--DNA adducts by the mammalian excision nuclease.

Nucleotide excision repair is one of the many cellular defense mechanisms against the toxic effects of cisplatin. An in vitro excision repair assay employing mammalian cell-free extracts was used to determine that the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA adduct, is excised by the excinuclease from a site-specifically modified oligonucleotide 156 base pairs in length. Repair of the minor interstrand d(G)/d(G) cross-link was not detected by using this system. Proteins containing the high mobility group (HMG) domain DNA-binding motif, in particular, rat HMG1 and a murine testis-specific HMG-domain protein, specifically inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occurred in a reconstituted system containing highly purified repair factors. These results indicate that HMG-domain proteins can block excision repair of the major cisplatin-DNA adducts and suggest that such an activity could contribute to the unique sensitivity of certain tumors to the drug. The reconstituted excinuclease was more efficient at excising the 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments using whole cell extracts [Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394-10398]. This result suggests that structural differences among the platinated DNA substrates, and not the presence of unidentified cellular factors, determine the relative excision repair rates of cisplatin-DNA intrastrand cross-links in the whole cell extracts.

Animals↗
PubMed1996

Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex.

cis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2[d(GpG)-N7(1), -N7(2)]] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by approximately 9 degrees C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25 degrees C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslink-induced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslink-induced perturbations might modulate protein binding.

Base Sequence↗
PubMed1996

Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo.

Ixr1 is a yeast HMG-domain protein that binds specifically to DNA adducts formed by the antitumor drug cisplatin. Interruption of the IXR1 gene in yeast desensitizes cells to cisplatin. This effect is unrelated to a natural function of Ixr1, which is to repress the transcription of COX5b. Ixr1 interacts specifically and preferentially with DNA modified by cisplatin. In the present work, Ixr1 was purified from a clone expressed in Escherichia coli. The dissociation constant for Ixr1 binding site-specifically to a 92-bp probe containing a single cis-[Pt(NH3)2{d(GpG)-N7(1) -N7(2)}] intrastrand cross-link was measured to be 2.5 (+/- 0.1) x 10(-7) M, similar to that found for HMG1. Ixr1 binds at least an order of magnitude more tightly to cisplatin-DNA adducts than to unmodified DNA. Hydroxyl radical footprinting revealed that Ixr1 protects an area of platinated DNA that is approximately 15 bp in size and centered at the platinum adduct. The binding of HMG-domain proteins to cisplatin-DNA adducts has been proposed to divert these proteins from their natural DNA-binding sites, disrupting transcription. This hypothesis was tested for Ixr1 in yeast. The protein was not titrated away from the Cox5b promoter sufficiently well to disrupt transcription either of Cox5b mRNA from genomic DNA or of the beta-galactosidase gene under control of the promoter in a plasmid DNA transformed into yeast.

Base Sequence↗
PubMed1996

Photoreactivity of platinum(II) in cisplatin-modified DNA affords specific cross-links to HMG domain proteins.

Cisplatin-modified DNA forms specific complexes with proteins that contain the DNA binding motif known as the high-mobility group (HMG) domain. As a tool for investigating the role of these proteins in mediating the cytotoxic effects of cisplatin, a set of cisplatin analogs was prepared in which one of the ammine ligands was replaced with a photoreactive tethered aryl azide ligand. The ability of DNA modified by these platinum complexes to photo-cross-link to HMG1 was investigated. During this study, it was discovered that DNA modified with cisplatin itself can undergo photoinduced cross-linking to HMG1 when irradiated with 300 nm light. The covalent complexes resulting from this latter cross-linking reaction are completely reversed by the addition of sodium cyanide and can be degraded by proteinase K. These results confirm the presence of a protein-DNA cross-link and demonstrate that the platinum atom itself forms the point of attachment. By contrast, DNA modified with transdiamminedichloroplatinum(II), [Pt(dien)Cl]Cl, or [Pt(NH3)3Cl]Cl does not cross-link to HMG1 upon irradiation. The photochemistry was exploited to cross-link a 15-base pair oligonucleotide containing a single, site-specific cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand adduct to domain B of HMG1. Following proteolytic digestion of the resulting covalent complex, the site of attachment to the protein was determined by Edman degradation of the resulting peptide-DNA complex to be a single residue on HMG domain B, Lys-6. The data further suggest that this amino acid binds to platinum at a site made available by photolabilization of a purine ligand. These results afford the first structural information about the interaction of HMG domain proteins with cisplatin-modified DNA.

Amino Acid Sequence↗
PubMed1996

The HMG-domain protein Ixr1 blocks excision repair of cisplatin-DNA adducts in yeast.

Ixr1 is a yeast HMG-domain protein which binds the major DNA adducts of the antitumor drug cisplatin. Previous work demonstrated that Saccharomyces cerevisiae cells lacking the IXR1 gene were two-fold less sensitive to cisplatin treatment than wild-type cells, and the present investigation reveals a six-fold difference in yeast having a different background. The possibility that the lower cytotoxicity of cisplatin in the ixr1 strain is the result of enhanced repair was investigated in rad1, rad2, rad4, rad6, rad9, rad10, rad14 and rad52 backgrounds. In three of the excision repair mutants, rad2, rad4 and rad14, the differential sensitivity caused by removing the Ixr1 protein was nearly abolished. This result demonstrates that the greater cisplatin resistance in the ixr1 strain is most likely a consequence of excision repair, supporting the theory that Ixr1 and other HMG-domain proteins can block repair of the major cisplatin-DNA adducts in vivo. The differential sensitivity of wild-type cells and those lacking Ixr1 persisted in the rad1 and rad10 strains, however, indicating that these two proteins act at a stage in the excision repair pathway where damage recognition is less critical. A model is proposed to account for these results, which is strongly supported recently identified functional roles for the rad excision repair gene products. A rad52 mutant was more sensitive to cisplatin than the RAD52 parental strain, which reveals that Rad52, a double-strand break repair protein, repairs cisplatin-DNA adducts, probably interstrand cross-links. A rad52 ixr1 strain was less sensitive to cisplatin than the rad52 IXR1 strain, consistent with Ixr1 not blocking repair of cisplatin adducts removed by Rad52 rad6 strains behaved similarly, except they were both substantially more sensitive to cisplatin. Interruption of the RAD9 gene, which is involved in DNA-damage-induced cell cycle arrest, had no affect on cisplatin cytotoxicity.

Antineoplastic Agents↗
PubMed1995

DNA adducts of cis-diamminedichloroplatinum(II) and its trans isomer inhibit RNA polymerase II differentially in vivo.

The effects of cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP adducts on mammalian transcription in vivo have been investigated. A plasmid containing the beta-galactosidase (beta-gal) reporter gene was modified with either of the two platinum compounds and transfected into human or hamster cell lines. A 2-3 fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans-DDP adducts was not the cause of its lower ability to block transcription in this assay. For this conclusion to be valid, it is assumed that trans-DDP adducts are repaired primarily by the nucleotide excision repair pathway, as is the case with the adducts of cis-DDP. The possibility that trans-DDP adducts are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of beta-gal mRNA on damaged templates in vivo. Nascent beta-gal mRNA transcripts were recovered from excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. Fourfold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63%. RNA polymerase II bypassed cis- and trans-DDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively. These data provide insight into the differential toxicity of the two platinum isomers.

Animals↗
PubMed1995

Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.

Bacteriophage M13↗
PubMed1995

Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin.

The success of cisplatin in cancer chemotherapy derives from its ability to crosslink DNA and alter the structure. Most cisplatin-DNA adducts are intrastrand d(GpG) and d(ApG) crosslinks, which unwind and bend the duplex to facilitate the binding of proteins that contain one or more high-mobility group (HMG) domains. When HMG-domain proteins such as HMG1, IXR (intrastrand-crosslink recognition) protein from yeast, or human upstream-binding factor (hUBF) bind cisplatin intrastrand crosslinks, they can be diverted from their natural binding sites on the genome and shield the adducts from excision repair. These activities sensitize cells to cisplatin and contribute to its cytotoxic properties. Crystallographic information about the structure of cisplatin-DNA adducts has been limited to short single-stranded deoxyoligonucleotides such as cis-[Pt(NH3)2(d(pGpG))]. Here we describe the X-ray structure at 2.6 A resolution of a double-stranded DNA dodecamer containing this adduct. Our information provides, to our knowledge, the first crystallographic look at a platinated DNA duplex and should help the design of new platinum and other metal crosslinking antitumour drug candidates. Moreover, the structure reveals a unique fusion of A- and B-type DNA segments that could be of more general importance.

Antineoplastic Agents↗
PubMed1995

A single HMG domain in high-mobility group 1 protein binds to DNAs as small as 20 base pairs containing the major cisplatin adduct.

Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.

Amino Acid Sequence↗
PubMed1995

Cisplatin and DNA repair in cancer chemotherapy.

Cisplatin, a DNA-damaging agent, is one of the most widely used anticancer drugs. As with all members of this class of chemotherapeutic compounds, the clinical success of cisplatin is compromised if tumor cells become resistant by various mechanisms, including enhanced DNA repair. In addition to its role in resistance, DNA repair has been linked to the cytotoxic mechanism of cisplatin. DNA damaged by the drug has proved to be a valuable tool for exploring the details of the nucleotide excision repair pathway.

Animals↗
PubMed1995

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states.

BACKGROUND: The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. RESULTS: The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. CONCLUSIONS: The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Binding Sites↗