Sequencing Gel Research Articles

Molecular Characterization of Campylobacter lanienae Strains Isolated from Food-Producing Animals

During 2008 and 2009, within the framework of the Hungarian monitoring program of antibiotic resistance of zoonotic agents from food-producing animals, a significant number (43 strains) of Campylobacter lanienae were detected for the first time in Hungary. The isolates were genotyped using partial 16S rRNA gene sequencing and pulsed-field gel electrophoresis using three different restriction enzymes. The antimicrobial resistance of the isolates was determined by microtiter broth dilution. C. lanienae isolation was successful only from swine but not from other animal species. According to phylogenetic analysis, clustering of the isolates shows the same extensive genetic diversity as other Campylobacter species. Sequence analysis of the partial 16S rRNA gene showed that additional variations exist in variable regions Vc2 and Vc6. SmaI restriction enzyme proved to be the most efficient for pulsed-field gel electrophoresis analysis of C. lanienae. A significant tetracycline resistance (60.9%) and the presence of erythromycin-, enrofloxacin-, and multiresistant C. lanienae strains were found. Although the pathogenic potential of C. lanienae in humans is currently unknown, this study demonstrates that C. lanieanae is common in pigs in the country, provides further details on the genotypic and phenotypic properties of C. lanienae, and offers a genotyping method for use in source tracing.

Perturbation of an arctic soil microbial community by metal nanoparticles

Technological advances allowing routine nanoparticle (NP) manufacture have enabled their use in electronic equipment, foods, clothing and medical devices. Although some NPs have antibacterial activity, little is known about their environmental impact and there is no information on the influence of NPs on soil in the possibly vulnerable ecosystems of polar regions. The potential toxicity of 0.066% silver, copper or silica NPs on a high latitude (>78°N) soil was determined using community level physiological profiles (CLPP), fatty acid methyl ester (FAME) assays and DNA analysis, including sequencing and denaturing gradient gel electrophoresis (DGGE). The results of these different investigations were amalgamated in order to develop a community toxicity indicator, which revealed that of the three NPs examined, silver NPs could be classified as highly toxic to these arctic consortia. Subsequent culture-based studies confirmed that one of the community-identified plant-associating bacteria, Bradyrhizobium canariense, appeared to have a marked sensitivity to silver NPs. Thus, NP contamination of arctic soils particularly by silver NPs is a concern and procedures for mitigation and remediation of such pollution should be a priority for investigation.

Isolation and characterization of methicillin-resistant Staphylococcus aureus from fire stations in two northwest fire districts

Methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated and characterized from environmental surfaces of two fire stations from two independent districts in the northwestern United States. After the first sampling and before the second sampling, education was provided, additional signage was added, and changes in disinfection protocols were put in place. Nasal carriage of MRSA was determined at the second sampling. Environmental samples were collected using SANICULT swabs and RODAC plates. Biochemical tests and 16S rRNA sequencing confirmed MRSA isolates. Antimicrobial susceptibility testing was performed, and the mecA gene, multilocus sequence typing, and SCCmec typing were determined by polymerase chain reaction, sequencing, and pulsed-field gel electrophoresis analysis. MRSA was isolated from 44 of 1,064 samples examined (4.1%) and included USA300 isolates. The same strains of MRSA were found in both the garage (ie, medic and fire trucks and protective clothing) and the living quarters. Nasal carriage of MRSA from one fire district was 22.5%. Community-like and hospital-like MRSA were isolated from the environmental samples. The majority of the nasal MRSA/S aureus isolates were genetically related to the environmental MRSA strains, suggesting possible transmission between personnel and the environmental surfaces. Further research is needed to verify this hypothesis.

Potassium Permanganate Probing of Pol II Open Complexes

INTRODUCTIONOne measure of an activator’s ability to stimulate preinitiation complex assembly is stimulation of the RNA polymerase II (Pol II) open complex. The reagent most commonly used to visualize open complexes is potassium permanganate (KMnO4). KMnO4 modifies thymine residues in single-stranded DNA but not in double-stranded DNA. The modified thymines are detected by primer extension via linear polymerase chain reaction (PCR). In the PCR, Taq polymerase stalls at the modified thymines on the template. Reactions are performed at 30°C and contain DNA template (typically, 10-50 ng present on a 4- to 5-kbp plasmid), nuclear extract, activator, and nonspecific carrier DNA. DNA melting by Pol II in vitro is energy dependent, so ATP is added to the reaction mix. After a brief 30-min incubation to permit assembly of the preinitiation complex, permanganate is added and the DNA is modified for a period of 1 min or more. The DNA is then purified and subjected to primer extension analysis; the products are fractionated as a series of bands on polyacrylamide/urea sequencing gels. Reactions with and without activator are compared, as are reactions with and without ATP. Titrations of the DNA template and activator and a time course are recommended.

Multifactorial Genesis of Pancreatitis in Primary Hyperparathyroidism: Evidence for “Protective” (PRSS2) and “Destructive” (CTRC) Genetic Factors

A relationship between primary hyperparathyroidism (pHPT) and pancreatitis has long been debated and remains a rare epiphenomenon. In a cohort of patients with pHPT and pancreatitis mutations in the serine protease inhibitor Kazal type I (SPINK1) and cystic fibrosis transmembrane conductance regulator (CFTR) genes, that increase the risk for pancreatitis have already been detected. Among the identification of additional pancreatitis-associtated mutations in the Chymotrypsin C gene (CTRC) it became clear that also protective genetic variants exist in the anionic trypsinogen gene (PRSS2) that decrease susceptibility for pancreatitis. Our aim was to detect either protective or inducing genetic factors in a large cohort of pHPT patients. Among 1,259 patients with pHPT, 57 patients were identified with pancreatitis (4.5%). DNA was available from 31 patients (16 acute pancreatitis/15 chronic pancreatitis). These individuals and 100 patients with pHPT without pancreatitis were analysed for CTRC (p.R254W and p.K247_R254del) and PRSS2 (p.G191R) mutations using melting curve analysis and DNA sequencing or PCR and gel electrophoresis (in case of p.K247_R254del CTRC). 2 of 31 patients with pHPT and pancreatitis carried the CTRC p.R254W missense mutation (6.5%), while all 100 pHPT controls without pancreatitis showed no CTRC mutation (P=0.055). No further SPINK1 p.N34S (n=4) mutations were detected but the probability of either CTRC or SPINK1 mutations in pHPT patients with pancreatitis is high (P<0.05). 1 patient was trans-heterozygous ( SPINK1: N34S/ CTRC p.R254W). CTRC p.K247_R254del was not detected in both groups. PRSS2 (p.G191R) mutation was present in 1 patient with pancreatitis (3.2%) and in 6 pHPT controls (6%) (P=1). This study underlines the relevance of a genetic background in pHPT related pancreatitis. However, it only indicates that the CTRC (p.R254W) mutation might also contribute to the panel of mutations ( SPINK1 and CFTR) that have been formerly reported to elevate pancreatitis susceptibility in pHPT. Besides it suggests that protective genetic variants, i. e., p.G191R PRSS2, may contribute to the low prevalence of pancreatitis in pHPT patients.

Unpaired 5′ ppp-Nucleotides, as Found in Arenavirus Double-stranded RNA Panhandles, Are Not Recognized by RIG-I

Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3' end (position +1). After formation of a dinucleotide, 5' pppGpC(OH) is then realigned on the template before this primer is extended. The net result of this "prime and realign" mechanism of genome initiation is that 5' pppG is found as an unpaired 5' nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5' pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5' nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5' ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.

Novel 12-bp deletion in the coding region of the bovineNPM1 gene affects growth traits

The nucleophosmin 1 gene (NPM1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in the control of various aspects of cell growth and homeostasis. In this study, the coding region of the NPM1 gene was screened in 1035 individuals of 4 Chinese cattle breeds by DNA sequencing and polyacrylamide gel electrophoresis. A novel 12-bp deletion mutation was identified in the coding region of the NPM1 gene. The PCR products of primer NPM1-P2 exhibited 3 genotypes and 2 alleles: 178 bp (denoted as W) and 166 bp (denoted as D). Genotype DD and allele D were predominant in the studied populations. Association analysis with growth traits in the Nanyang breed (N = 265) showed that the animals with genotype DD had significantly greater birth weight, body weight, body length, and heart girth than those with genotype WD (P < 0.01 or P < 0.05) at birth and after 6 months and 12 months, but not at 18 and 24 months of age. Results of this study suggest that the NPM1 gene is a candidate gene for growth traits in cattle.

Seasonal variation in nutrient limitation of microbial biofilms colonizing organic and inorganic substrata in streams

Humans have increased the availability of nutrients including nitrogen and phosphorus worldwide; therefore, understanding how microbes process nutrients is critical for environmental conservation. We examined nutrient limitation of biofilms colonizing inorganic (fritted glass) and organic (cellulose sponge) substrata in spring, summer, and autumn in three streams in Michigan, USA. Biofilms were enriched with nitrate (NO3 −), phosphate (PO4 3−), ammonium (NH4 +), NO3 − + PO4 3−, NH4 + + PO4 3−, or none (control). We quantified biofilm structure and function as chlorophyll a (i.e., primary producer biomass) and community respiration on all substrata. In one stream, we characterized bacterial and fungal communities on cellulose in autumn using clone library sequencing and denaturing gradient gel electrophoresis to determine if community structure was linked to nutrient limitation status. Despite oligotrophic conditions, primary producer biomass was infrequently nutrient limited. In contrast, respiration on organic substrata was frequently limited by N + P combinations. We found no difference between biofilm response to NH4 + versus NO3 − enrichment, although the response to both N-species was positively related to water column PO4 3− concentrations and temperature. Molecular analysis for fungal community composition suggested no relationship to nutrient limitation, but the dominant members of the bacterial community on cellulose were different on NO3 −, PO 4 3 , and NO3 − + PO4 3− treatments relative to control, NH4 +, and NH4 + + PO4 3− treatments, which matched patterns for biofilm respiration rates from each treatment. Our results show discrete patterns of nutrient limitation dependent upon substratum type and season, and imply changes in bacterial community structure and function may be linked following nutrient enrichment in streams.

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase lambda (Pol lambda) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol lambda. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol lambda efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol lambda could modulate the cytotoxic effect of AraC.

Characterization of Enterococcus spp. from Human and Animal Feces Using 16S rRNA Sequences, theespGene, and PFGE for Microbial Source Tracking in Korea

Contamination from human and animal fecal waste is a primary cause of water pollution. Microbial source tracking (MST) may be a useful tool for high-quality environmental management and for assessing human health risks associated with water pollution. The goal of this study was to evaluate Enterococcus spp. as a target organism for MST. Thirty-four fecal samples were collected from five different sources (human, chicken, pig, cow, and goose) in South Korea. In total, 237 Enterococcus spp. were isolated from feces using membrane- Enterococcus indoxyl-beta-d-glucoside agar. The 16S rRNA gene and the whole genome were analyzed using nucleic acid sequencing and pulsed-field gel electrophoresis (PFGE), respectively. Both phylogenetic analysis and principal coordinate analysis using UniFrac were performed on the nucleic acid sequences of the 16S rRNA gene. According to P-tests from UniFrac, significant differences existed between Enterococcus spp. isolated from human feces and those from animal feces. In addition, we evaluated whether the esp gene of Enterococcus faecium could be a specific target for Enterococcus spp. isolated from human feces. Of 58 E. faecium isolates tested, only three were esp-positive. The specificity of the esp gene of E. faecium isolated from human feces was 100%, but the sensitivity was <10%. These results suggest that Enterococcus spp. have different molecular characteristics according to their fecal source and that these characteristics can be further identified by analyzing the esp gene and 16S rRNA sequences, whereas PFGE provides limited information on the fecal sources of Enterococcus spp.

Direct Interaction between Quorum-sensing Regulator SmcR and RNA Polymerase Is Mediated by Integration Host Factor to Activate vvpE Encoding Elastase in Vibrio vulnificus

It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.

Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

Thiolated tRNAs of Trypanosoma brucei Are Imported into Mitochondria and Dethiolated after Import

All mitochondrial tRNAs in Trypanosoma brucei derive from cytosolic tRNAs that are in part imported into mitochondria. Some trypanosomal tRNAs are thiolated in a compartment-specific manner. We have identified three proteins required for the thio modification of cytosolic tRNA(Gln), tRNA(Glu), and tRNA(Lys). RNA interference-mediated ablation of these proteins results in the cytosolic accumulation non-thio-modified tRNAs but does not increase their import. Moreover, in vitro import experiments showed that both thio-modified and non-thio-modified tRNA(Glu) can efficiently be imported into mitochondria. These results indicate that unlike previously suggested the cytosol-specific thio modifications do not function as antideterminants for mitochondrial tRNA import. Consistent with these results we showed by using inducible expression of a tagged tRNA(Glu) that it is mainly the thiolated form that is imported in vivo. Unexpectedly, the imported tRNA becomes dethiolated after import, which explains why the non-thiolated form is enriched in mitochondria. Finally, we have identified two genes required for thiolation of imported tRNA(Trp) whose wobble nucleotide is subject to mitochondrial C to U editing. Interestingly, down-regulation of thiolation resulted in an increase of edited tRNA(Trp) but did not affect growth.

Efficient photooxidative strand cleavage at 5-methylcytosine in DNA by sensitization with 9,10-anthraquinone-tethered oligonucleotides

Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of 5-methylcytosine ((m)C)-containing DNA and 9,10-anthraquinone (AQ)-tethered complementary oligodeoxynucleotide led to selective oxidative strand cleavage at the target (m)C site, whereas no strand cleavage was observed for the duplex containing normal cytosine instead of (m)C. Incorporation of an AQ sensitizer into the interior of a strand induced an enhanced one-electron photooxidation, presumably because of a much larger intersystem crossing efficiency, leading to an efficient strand cleavage at the target (m)C site in DNA. Optimization of the photosensitizer could afford more strand cleavage at (m)C in DNA, thereby allowing for the more sensitive detection of the target (m)C on a sequencing gel.

The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon β

Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.

DiaA Dynamics Are Coupled with Changes in Initial Origin Complexes Leading to Helicase Loading

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg(404), Lys(408), Tyr(411), and Gly(415)) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr(411) and Gly(415) are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp(263) located in the linker of one subunit can interact with Tyr(411), Lys(408), or Arg(404) in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp(263) and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.

Association between entero‐hepatic Helicobacter species and Crohn’s disease: a prospective cross‐sectional study

The pathogenesis of Crohn's disease (CD) involved microbial factors. Some Helicobacter species, the so-called entero-hepatic Helicobacters (EHH), can naturally colonize the intestinal surface and have been detected in humans. Aim To look for an association between CD and the presence of EHH DNA in intestinal biopsies. Two groups of patients were included prospectively in a multicentre cross-sectional study: CD patients with an endoscopic post-operative recurrence within 2 years following a surgical resection and controls screened for colorectal polyps or cancer. Intestinal biopsies were taken for Helicobacter culture and Helicobacter 16S DNA detection. If positive, the EHH species were identified with specific PCRs, sequencing and denaturing gradient gel electrophoresis. In the 165 included patients (73 CD and 92 controls), Helicobacter cultures were negative. PCR was positive in 44% of CD and 47% of controls. After age-adjustment, CD was significantly associated with EHH in intestinal biopsies (OR = 2.58; 95%CI: 1.04-6.67). All EHH species detected were identified as Helicobacter pullorum and the closely related species Helicobacter canadensis. Crohn's disease is associated with the presence of EHH species DNA in intestinal biopsies after adjustment for age. Whether these species play a role in the pathophysiology of CD remains to be determined.

Cisplatin-damaged BRCA1 exhibits altered thermostability and transcriptional transactivation

BRCA1 is a tumor suppressor gene. Its translated product has an important function in transcriptional activation and DNA repair pathways. Damaged BRCA1 due to cisplatin treatment may lead to loss of such functions. To address a potential drug target of BRCA1 for cisplatin treatment, we investigated the biophysical characterization and functional consequences of the 3/-terminal region of human BRCA1 after in vitro platination with cisplatin. To analyze the base/sequence specificity of cisplatin damage, the measurement for sensitivity of cisplatin-treated BRCA1 to restriction enzymes (EcoO109I and PvuII) and sequence gel analysis was conducted. The results suggested that the platination favorably occurred at the d(GpG) and the d(GpC) sites. An increase in drug concentrations resulted in increased interstrand crosslinks at the d(GpC) site. Cisplatin affected the transition temperature of the BRCA1 gene fragment in a biphasic fashion. DSC thermogram of DNA adducts was shifted to a lower transition temperature at lower cisplatin concentration. However, at higher drug concentration, the thermogram peaked at a slightly higher transition temperature with predominantly increased heat specific capacity. Reduction in cellular DNA repair of cisplatin-damaged plasmid DNA, using host cell reactivation assay, was a consequence of an increase in platination levels on the reporter gene. The GAL4-fused BRCA1 slightly enhanced the functional consequences of cisplatin-BRCA1 adducts 3 transcription of the reporter gene in the absence of GAL4 binding site. The transcriptional transactivation activity of cisplatin-modified BRCA1, when tested in “one-hybrid GAL4 transcriptional assay”, was inversely proportional to cisplatin doses. Furthermore, the transcriptional transactivation activity was dramatically diminished in the presence of a second expression vector containing multiple cisplatin-damaged sites. The data provide the first evidence for direct interaction of cisplatin with BRCA1 and raise the possibility of BRCA1 as a therapeutic target for platinum drug- based chemotherapy.