Cisplatin binds to pre-miR-200b and impairs its processing to mature microRNA.

Cisplatin is an important anticancer drug with a complex mode of action, a variety of possible targets, and numerous resistance mechanisms. While genomic DNA has traditionally been considered to be its most critical anticancer target, several lines of evidence suggest that various RNAs and other biomolecules may play a role in its anticancer mode of action. In this report we demonstrate that cisplatin modifies pre-miR-200b, impairs its processing to mature miRNA, and decreases miR-200b expression in ovarian cancer cells. Considering the role of miR-200b in epithelial-to-mesenchymal transition and cancer chemosensitivity, cisplatin-induced modification of pre-miR-200b and subsequent deregulation of mature miR-200b may, depending on cell context, limit anticancer activity of this important anticancer drug. More gener- ally, precursor miRNAs may be important targets of cisplatin and play a role in this drug's anticancer activity or modulate cell responses to this drug.

Cloning DNA restriction endonuclease fragments with protruding single-stranded ends.

A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C).

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.

DNA bending induced by the catabolite activator protein allows ring formation of a 144 bp DNA.

The effect of the catabolite activator protein, CAP, on the ligation of a 144 bp DNA was examined. This DNA has EcoRI ends and contains the lac operon CAP site and promoter-operator region. At low DNA concentrations (nM) and 37 degrees C the presence of CAP and cAMP enables T4 ligase to convert the linear duplex to a covalently closed ring. Nuclease digestion and sedimentation equilibrium studies show that the ring is a monomer circle. Ring formation does not occur in the absence of either CAP or cAMP. The kinetics of ring closure, and the bimolecular joining of two fragments were measured. The presence of CAP decreased the rate of bimolecular joining of the EcoRI ends of linear DNAs. Thus the measured rates of ring closure are likely to be a lower limit for this process. Closure reactions carried out with ethidium bromide indicate that CAP induced bending rather than twisting is responsible for ring formation. The all or none nature of the closure reaction suggests that persistence length DNAs may be useful in a simple assay for protein induced DNA bending.

Raman spectroscopy study of the B-Z transition in (dG-dC)n.(dG-dC)n and a DNA restriction fragment.

The B to Z conformational transition of (dG-dC)n.(dG-dC)n and a 157 bp DNA restriction fragment were followed using Raman spectroscopy. The 157 bp DNA has a 95 bp segment from the E. coli lactose operon sandwiched between 26 and 32 bp of (dC-dG) sequences. Raman spectra of the DNAs were obtained at varying sodium chloride concentrations through the region of the transition. A data analysis procedure was developed to subtract the background curves and quantify Raman vibrational bands. Profiles of relative intensity vs. sodium chloride concentration are shown for bands at 626, 682, 831-833 and 1093 cm-1. Both (dG-dC)n.(dG-dC)n and the 157 bp DNA show changes in the guanine vibration at 682 cm-1 and backbone band at 831-3 cm-1 preceding a highly cooperative change in the 1093 cm-1 PO2- vibration. This result indicates that there are at least two conformational steps in the B to Z conformational pathway. We review the effect of the (dC-dG) portion of the 157 bp DNA on the 95 bp segment. Comparison of Raman spectra of the 157 bp DNA, the 95 bp fragment and (dG-dC)n.(dG-dC)n indicate that in 4.5 M NaCl the (dC-dG) segments are in a Z-conformation. Base stacking in the 95 bp portion of the 157 bp DNA appears to maintain a B-type conformation. However, a substantial portion of this region no longer has a B-type backbone vibration.

Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods.

Temperature gradient gel electrophoresis (TGGE) and related methods can separate DNA fragments that differ by a single base pair or defect. This article describes the basic features of TGGE, and reviews the theoretical model of DNA unwinding and its ability to predict DNA mobility in a temperature gradient gel. Recent applications of TGGE and related methods that were directed at detecting point mutations, and evaluating the effects of single site defects are also reported.

Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40.

The Escherichia coli strain FC40 has frequently been employed to investigate the mechanism of adaptive mutations. The strain cannot utilize lactose due to a +1 frameshift mutation that reduces beta-galactosidase to about 1% of normal levels. Cells undergo a high rate of mutation from Lac- to Lac+ when cells are grown with lactose as the sole energy source. Almost all Lac+ colonies arising 3-6 days after plating result from a base pair deletion in runs of iterated base pairs within a 130-bp target region. In this study we characterized Lac+ colonies arising 3-10 days after plating. Temperature gradient gel electrophoresis (TGGE) was used to detect mutations in the target region as a function of the day a colony appears. TGGE results confirmed the occurrence of mutations within the target region in 36 of 37 FC40 Lac+ colonies arising on days 3-7. However, mutations in this region were not detected in 23 of 37 Lac+ colonies arising from days 8-10. Sequencing data verified the TGGE results. Half of the Lac+ mutants arising on days 8-10 with no base pair change in the target region were unstable and exhibited a Lac- phenotype after successive growth cycles in rich medium. The results suggest that amplification of the lac operon region is a common factor in late arising colonies, and that different characteristics distinguish early and late arising Lac+ colonies.

The transmission of stability or instability from site specific protein-DNA complexes.

Theoretical calculations were made to determine the influence of side specific 'melting' and 'stabilizing' proteins on the thermal stability of nearby base pairs (bp). A DNA sequence 999bp. long containing the 123 bp. lactose operon control region in the center was examined. Melting curves of base pairs near the binding sites of the catabolite activator protein, CAP, the lactose repressor, and RNA polymerase were calculated in the absence and presence of each protein. The empirical loop entropy model of the helix-coil transition of DNA was employed. Calculations show that melting and stabilizing proteins alter the tm of base pairs 20 to 100 bp-away. The magnitude and range of the effect is strongly influenced by the base pair composition and sequence of the protein site and the immediately adjacent DNA regions.

DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry.

Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.

Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters.

The regions upstream from forty-three procaryotic promoters were examined for nucleotide distributions which have been associated with DNA curvature. The analysis procedure assigned a DNA curvature score based on the phasing of the 5' and 3' ends of An and Tn tracts, n greater than or equal to 3. The weighting scheme for the curvature score was based on recent studies which showed that tracts of An and Tn periodically phased with the helix repeat cause DNA curvature. Results show that promoters which have high transcription initiation rates in vivo tend to have high curvature scores in their upstream regions. Regions downstream from the transcription start-point do not have sequences correlated with DNA curvature. Four promoters which have been shown to have upstream activation regions have curvature scores above 1.5 in their -40 to -150 regions. The correlations observed lend support to the hypothesis that DNA curvature is associated with upstream activation of transcription.

The relative stabilities of base pair stacking interactions and single mismatches in long RNA measured by temperature gradient gel electrophoresis.

The thermal stability of RNA duplexes differing by a single base pair (bp) substitution or mismatch were investigated by temperature gradient gel electrophoresis (TGGE). All base pair substitutions and mismatches were examined at six sites, and limited changes were investigated at three other sites. DNA templates for in vitro transcription were generated by the polymerase chain reaction (PCR). Transcribed forward and reverse single stranded RNAs were annealed to form 345 bp dupex RNA. Solution melting curves of selected RNAs were in good agreement with the predicted three step transitions. Parallel TGGE was used to determine the relative stabilities of the RNAs, and perpendicular TGGE was employed to obtain mobility transitions and midpoint transition temperatures (Tmu) of the RNAs' first melting domain. The gel solvent included formamide and urea. The Tmu values of the first melting domain were influenced by the identity of the base pair substitution or mismatch as well as by the site's neighboring base pairs. The difference in the transition temperatures (deltaTmu) between pairs of RNA ranged from 0 to 5 degrees C. deltaTmu values were used to determine free energy differences (deltaDeltaG). For RNA pairs distinguished by a base pair substitution, the deltaDeltaG values were closely correlated with free energy differences calculated from stacking free energies determined from melting studies in 1 M Na+ [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261.] An algorithm was developed using the free energies of terminal mismatches [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261] that provided very good agreement with experimental free energies for the single internal mismatches.

Characteristics and variations of B-type DNA conformations in solution: a quantitative analysis of Raman band intensities of eight DNAs.

Raman spectra were obtained from four bacterial DNAs varying in GC content and four periodic DNA polymers in 0.1 M NaCl at 25 degrees C. A curve fitting procedure was employed to quantify and compare Raman band characteristics (peak location, height, and width) from 400 to 1600 cm-1. This procedure enabled us to determine the minimum number of Raman bands in regions with overlapping peaks. Quantitative comparison of the Raman bands of the eight DNAs provided several new results. All of the DNAs examined required bands near 809 (+/- 7) and 835 (+/- 5) cm-1 to accurately reproduce the experimental spectra. Since bands at these frequencies are associated with A-family and B-family conformations, respectively, this result indicates that all DNAs in solution have a mixture of conformations on the time scale of the Raman scattering process. Band characteristics in the 800-850-cm-1 region exhibited some dependence on CG content and base pair sequence. As previously noted by Thomas and Peticolas [Thomas, G. A., & Peticolas, W. L. (1983) J. Am. Chem. Soc. 105, 993], the poly[d(A)].poly[d(T)] spectra were qualitatively distinct in this region. The A-family band is clearly observed at 816 cm-1. The intensity of this band and that of the B-family band at 841 cm-1 were similar, however, to intensities in the natural DNA spectra. Three bands at 811, 823, and 841 cm-1 were required to reproduce the 800-850-cm-1 region of the poly[d(A-T)].poly[d(A-T)] spectra. This may indicate the presence of three backbone conformations in this DNA polymer. Analysis of intensity vs. GC content for 42 Raman bands confirmed previous assignments of base and backbone vibrations and provided additional information on a number of bands.

The effect of base sequence on the stability of RNA and DNA single base bulges.

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.

Influence of neighboring base pairs on the stability of single base bulges and base pairs in a DNA fragment.

Temperature-gradient gel electrophoresis (TGGE) was used to determine the relative thermal stabilities of 32 DNA fragments that differ by a single unpaired base (base bulge) and 17 DNAs differing by a base pair. Homologus 373 and 372 bp DNA fragments differing by a single base pair substitution or deletion were employed. Heteroduplexes containing a single base bulge were formed by melting and reannealing pairs of 372 and 373 bp DNAs. Product DNAs were separated on the basis of their thermal stability by parallel and perpendicular TGGE. The order of stability was determined for all single unpaired bases in four different nearest neighbor environments: (GXT).(AYC), (GXG).(CYC), (CXA).(TYG), and (TXT).(AYA) with X = A, T, G, or C, and Y = no base, or visa versa. DNA fragments containing a base bulge were destabilized by 2-3.6 degrees C (+/- 0.2 degrees C) with respect to homologous DNAs with complete Watson-Crick base pairing. Both the identity of the unpaired base and the sequence of the flanking base pairs influenced the degree of destabilization. The range of temperature shift correspond to estimated unfavorable free energies from 2.5 to 4.6 kcal/mol. Purine base bulges were generally not as destabilizing as pyrimidine base bulges. An unpaired base which was identical to one of its adjacent bases generally caused less destabilization than an unpaired base with an identity differing from its nearest neighbors. This implies that positional degeneracy of an unpaired base within a run of two or more identical bases is an important factor effecting stability.(ABSTRACT TRUNCATED AT 250 WORDS)

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site.

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.

A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods.

A computer program was developed to automate the selection of DNA fragments for detecting mutations within a long DNA sequence by denaturing gel electrophoresis methods. The program, MELTSCAN, scans through a user specified DNA sequence calculating the melting behavior of overlapping DNA fragments covering the sequence. Melting characteristics of the fragments are analyzed to determine the best fragment for detecting mutations at each base pair position in the sequence. The calculation also determines the optimal fragment for detecting mutations within a user specified mutational hot spot region. The program is built around the statistical mechanical model of the DNA melting transition. The optimal fragment for a given position is selected using the criteria that its melting curve has at least two steps, the base pair position is in the fragment's lowest melting domain, and the melting domain has the smallest number of base pairs among fragments that meet the first two criteria. The program predicted fragments for detecting mutations in the cDNA and genomic DNA of the human p53 gene.

RNase H1 can catalyze RNA/DNA hybrid formation and cleavage with stable hairpin or duplex DNA oligomers.

Cleavage of a RNA target site by RNase H1 from Escherichia coli was examined in the presence of complementary DNA sequences in the form of single-stranded, duplex, and hairpin structures. The target site was a 15 nt sequence in the middle of a 79 nt RNA transcript. DNA molecules employed included seven single-stranded oligodeoxynucleotides 10 or 15 nt long, and five hairpin DNAs each with a 10 bp stem and 5 nt loop. The loop and 3' side of the stem of two of the hairpin DNAs were fully complementary to the target site, while the other hairpin DNAs had sequence changes. A 10 bp duplex DNA with one strand complementary to the target site was also employed. A gel electrophoresis mobility shift assay examined hybrid formation between the RNA and the single-stranded 15 nt DNA and two hairpin DNAs that contained 15 complementary bases. RNA titration of the 32P-labeled single-stranded DNA produced a shifted band indicative of RNA/DNA complex formation. No RNA/DNA complex was detected when the more stable (Tm = 71 degrees C) hairpin DNA was combined with excess RNA. The less stable hairpin DNA (Tm = 62 degrees C) showed a small amount ( approximately 8%) of hybrid formation. Thermodynamic analysis of RNA binding to the DNAs was in qualitative agreement with the results. Although no RNA/DNA hybrid was expected from thermodynamic calculations, a RNase H assay at 25 degrees C showed that hairpin or duplex DNAs with a 10 nt complementary sequence catalyzed RNA degradation. A complementary loop sequence in the hairpin DNA was not required. Cleavage of the RNA did not occur with hairpin DNAs containing three or four noncomplementary bases in the stem. The results show that RNase H can promote the formation and cleavage of a RNA/DNA hybrid between an RNA site and a base paired strand of a stable hairpin or duplex DNA at temperatures below their Tm.

Inverted repeat sequences can influence the melting transitions of linear DNAs.

The influence of inverted repeat sequences on the melting transitions of linear DNAs has been examined. Derivative melting curves (DMC) of a 514 base pair (bp) DNA, seven subfragments of this DNA, and four other DNAs have been compared to predictions of DNA melting theory. The 514-bp DNA contains three inverted repeat sequences that can form cruciform structures in supercoiled DNA. We refer to these sequences as c-inverted repeats. Previous work showed that the DMC of this DNA, unlike a number of other DNAs, is not accurately predicted by DNA melting theory. Since the theoretical model does not include hairpin-like structures, it was suggested that hairpin or cruciform formation in these inverted repeats may be responsible for this discrepancy. Our results support this hypothesis. Predicted DMCs are in good agreement with DNAs with no inverted repeats, or inverted repeats not evident in supercoiled DNA. Differences between the theoretical and experimental Tm's are less than or equal to 0.3 degrees C. DNA molecules that contain one or more of the three c-inverted repeats are not as accurately predicted. Experimental Tm values are lower than predicted values by 0.7-3.8 degrees C. It is concluded that some inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. These structures appear to lower overall DNA stability.

Theory agrees with experimental thermal denaturation of short DNA restriction fragments.

Experimental melting transitions of several natural DNAs of known nucleotide sequences have recently been obtained. The differential melting curves of these DNAs-phi X174 DNA, fd DNA and SV40 DNA-all show distinctive sets of peaks or fine structure. Theoretical melting curves calculated from the sequences and a few a priori parameters have not accurately predicted the experimental transitions. Although calculated fine structure resembled experimental curves in some cases, the characteristic features of a DNA's differential melting curve could not generally be produced. Azbel and Gabbarro-Arpa et al. have recently obtained good agreement between calculated and experimental curves using a different theoretical approach-only ground-state configurations of DNA were considered for temperatures inside the transition region. Their results suggest that the basic model of DNA melting, common to all theoretical approaches, is accurate. We have used here an exact theoretical approach to calculated melting curves of four DNA restriction fragments of 95-301 base pairs containing the lactose promoter region (Fig. 1). Theoretical curves agree very well with the experimental transitions published by Hardies et al. and obtained in this laboratory.