Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Melting of cross-linked DNA IV. Methods for computer modeling of total influence on DNA melting of monofunctional adducts, intrastrand and interstrand cross-links formed by molecules of an antitumor drug

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Gel electrophoresis of partially denatured DNA. Retardation effect: its analysis and application.

The hypothesis about the role of partial denaturation in DNA retardation during its electrophoresis in denaturing gel /1,2/ was tested. We used partially melted DNA molecules in which the size of the melted regions and their location were known. They were obtained through glyoxal treatment of the melted regions by a procedure allowing the denatured state to be fixed at any point within the melting range. The approach and the availability of the melting maps of DNAs made it possible to investigate DNA molecules differing in length and in the size of the melted regions. The presence of a denatured region at the end of the molecule or inside of it was shown to decrease its electrophoretic mobility, the effect depending on the size of the melted region and on the DNA length. On the basis of the experimental results an explanation is proposed for the cause of retardation in the case of partially denatured DNA.     

Role of small loops in DNA melting

Short melted regions less than 100 base pairs (bp) in length are rarely found in the differential melting curves (DMC) of natural DNAs. Therefore, it is supposed that their characteristics do not affect DNA melting behavior. However, in our previous study, a strong influence of the form of the entropy factor of small loops on melting of cross-linked DNAs was established (D. Y. Lando, A. S. Fridman et al., Journal of Biomolecular Structure and Dynamics, 1997, Vol. 15, pp. 141-150; Journal of Biomolecular Structure and Dynamics, 1998, Vol. 16, pp. 59-67). Quite different dependencies of the melting temperature on the relative concentration of interstrand cross-links were obtained for the loop entropy factors given by the Fixman-Freire (Jacobson-Stockmayer) and Wartell-Benight relations. In the present study, the influence of the entropy factor of small loops on the melting of natural DNAs, cross-linked DNAs and periodical double-stranded polynucleotides is compared using computer simulation. A fast combined computational method for calculating DNA melting curves was developed for this investigation. It allows us to assign an arbitrary dependence of the loop entropy factor on the length of melted regions for the terms corresponding to small loops (less than tau bp in length). These terms are calculated using Poland's approach. The Fixman-Freire approach is used for long loops. Our calculations have shown that the temperature dependence of the average length of interior melted regions (loops) has a maximum at T approximately T(m) (T(m) is the DNA melting temperature) in contrast to the dependence of the total average length of melted regions, which increases almost monotonously. Computer modeling demonstrates that prohibition of formation of loops less than tau base pairs in length does not markedly change the DMC for tau < 150 bp. However, the same prohibition strongly affects the average length of internal melted regions for much smaller tau's. The effect is already noticeable for tau = 1 bp and increases with tau. A tenfold increase in the entropy factor of all loops with length less than tau bp causes a noticeable alteration of the DMC for tau > or = 30 bp. It is shown that DMCs are identical for the Wartell-Benight and for the Fixman-Freire (Jacobson-Stockmayer) form of the loop entropy factor. However, for low degree of denaturation, the average length of internal melted regions is 40% lower for the Wartell-Benight form due to the fluctuational opening of short AT-rich regions less than 10 bp in length. The same calculations carried out for periodical polynucleotides demonstrate a much stronger difference in melting behavior for different forms of entropy factors of short loops. The strongest difference occurs if the length of stable GC-rich and unstable AT-rich stretches is equal to 30 bp. However, the comparison carried out in this work demonstrates that the entropy factor of short loops influences melting behavior of cross-linked DNA much stronger than of unmodified DNA with random or periodical sequences.     

Catalysis of strand annealing by replication protein A derives from its strand melting properties

Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability.     

Mechanism of DNA denaturation in the presence of manganese ions

The unwinding of DNA strands in the presence of small concentrations of Mn²⁺ ions (2 × 10^?4?4 × 10^?4M) has been studied. The process of unwinding is nonequilibrium; the DNA strands are gradually unwound at a constant temperature corresponding to the beginning of the melting curve. There is no true renaturation in the partially melted DNA. It is shown in the paper that these effects are due to the aggregation of the unwound DNA regions. The Mn²⁺ ions are responsible for the binding of the unwound strands. The aggregation precludes renaturation, shifts the equilibrium towards the melted state, and causes slow unwinding at a constant temperature. The binding of denaturated regions seems to occur through the guanines.     

Repeated sequences in the DNA of the eukaryotic genome

Long DNA molecules from a cucumber satellite, the cucumber main band, mung bean, and Chinese hamster ovary (CHO) were digested with mung bean nuclease I, which was used as a probe for high AT regions. The digests were viewed under the electron microscope, and the distribution of sizes for the fragments of nuclease-treated plant DNA showed that the main band cucumber and the mung bean have regions along their genomes spaced at approximately 0.3 to 0.4 μ that are sensitive to the nuclease. The satellite from the cucumber contains these sites at intervals generally of 0.1 μ or less, whereas CHO DNA has these regions at intervals of 0.05 to 1.40 μ in length. The long DNA from the main band of the cucumber and the CHO were also partially melted in formamide at 37°C to denature preferentially the regions along the DNA molecules that are rich in AT. Measurements of the distances from the center of each loop to the center of the adjacent loops showed that these distances for the main band cucumber DNA tended to occur at approximately every 0.4 μ, whereas the corresponding distances for the Chinese hamster DNA were less regular, occurring every 0.1 to 1.0 μ.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in Mus musculus DNA were examined by electron microscopy (EM). The majority (85%) of palindromes were found to be clustered in about one half of the DNA strands. The rest of the DNA strands were seen with a solitary looped structure. — The unlooped palindromes constituted 53% of all palindromes and were always clustered. There was a significant reduction in the number of unlooped palindromes in comparison to D. melanogaster DNA (Biezunski, 1981) and as a result the palindrome clusters were smaller and contained 2–8 palindromes [4–16 inverted repeats (ir)] per DNA strand. The looped palindromes had a wide and regular distribution with spacing lengths similar to those found in D. melanogaster DNA, and showed some periodicity. The average spacing between centers of all palindromes (inside a cluster) was 4.325 kb, and between centers of looped palindromes 8.544 kb. — The lengths of the ir of unlooped and looped palindromes were grouped (similar to D. melanogaster DNA) in one size-class with a range of 30–240 bp and an average length of 130 bp. Longer ir were also observed and the average length of ir in unlooped palindromes was 186 bp, in looped 588 bp, and the total average length was 375 bp. — It was calculated that there are about 224,000–320,000 palindromes (ir pairs) in the mouse genome, with the spacing between centers of all palindromes about 13-9 kb in length. — In high molecular weight mouse DNA, complex looped structures composed of rows of 5–8 looped palindromes one on top the other, formed by renaturation of multiple ir, were observed. It is suggested, that clustered repetitive sequences, in direct and inverted orientation, might be of one family and homologous to one another, and be able to reassociate, in vitro and in vivo, into structures of different forms, which could function as binding sites for various regulatory proteins during mouse development.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

The organization of DNA segments undergoing repair synthesis during pachytene

DNA regions undergoing programmed repair synthesis during pachytene were isolated and used as a probe for analyzing the organization of these regions. Segments that are the sites of nick-repair activity are referred to as PsnDNA. These segments are distributed at intervals ranging from 30–350 kilobase pairs (kbp) within about half the genome. The other half of the genome, which consists of DNA molecules longer than 350 kbp under defined conditions of extraction, lacks these segments. PsnDNA sequences range in length from about 150–300 base pairs (bp) and are arranged in larger P.DNA units measuring 0.8–3.0 kbp. P.DNA units have three identifiable regions. Each end region consists of a PsnDNA sequence and the middle region contains sequences that do not share homology with PsnDNA and have a much lower repeat number. Pachytene nicking of PsnDNA sequences is polar with respect to the orientation of individual DNA strands. Most of the PsnDNA sequences are present at the 5 ends of the single strands generated in vivo by endonuclease action. Nicking is probably repeated at each PsnDNA site during early and midpachytene, and both members of a duplex are nicked within any single P.DNA region.     

Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Overstretching of DNA occurs at about 60–70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.     

Spontaneous sharp bending of DNA: role of melting bubbles

The role of centrally located and distributed base pair mismatches (‘melting bubbles’) on localized bending and stiffness of short dsDNA fragments is evaluated using time-dependent fluorescence lifetime measurements. Distributed melting bubbles are found to induce larger bending angles and decreased levels of stiffness in DNA than centrally located ones of comparable overall size. Our results indicate that spontaneous local opening-up of the DNA duplex could facilitate sharp bending of short DNA strands even in the absence of DNA binding proteins. We also find that the occurrence of two closely spaced melting bubbles will generally be favored when a large energetic barrier must be overcome in forming the desired bent DNA structure.     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary A comparison of the tight-junctions of various cell types in the nasal epithelia of frog, ox, rat and dog shows that Bowman's gland cells have lowest number of strands (4–8), whereas olfactory receptor and supporting, and ciliated respiratory cells show no conspicuous differences and have 6–11 strands. Tight-junctional strand numbers show slight species-dependent variations. In regions where three cells join (observed for receptor and respiratory cells), fracture faces show two parallel strands which fuse at certain points. These strands run perpendicularly to the rest of the tight-junctional belt, which also shows an increased number of strands (13–16) in this region.Tight-junctions of mammalian olfactory dendritic endings usually show strands composed of particles, whereas those of the other three epithelial cell types consist of continuous or discontinuous bars. Tight-junctions of dendritic endings of the frog also conform to the latter type. Differences in strand density are only slight and range from 16–27 strands/m. Small angular gap-junctions were observed only within the tight-junctions of supporting cells in the rat.     

Substructure of thermally unfolded chromatin subunits or nucleosomes

Mononucleosomes containing 143 ± 6 base pairs of duplex DNA and approximately two copies each of the histones H2a, H2b, H3 and H4 were examined during thermal denaturation by high resolution electron microscopy using both bright- and dark-field (tilted beam) modes. Co-operative destabilization and unfolding of the 13.2 ± 1.4 nm diameter toroids occurred only after the second of the two major melting transitions. The unfolding patterns are consistent with about 1.5–1.8 turns of supercoiled DNA in intact nucleosomes. The dominant unfolded structure of samples post-fixed with glutaraldehyde is a 17.5 ± 2.1 nm diameter open ring. Both sister DNA strands remain associated with protein. The distribution and shape of the protein patches are more irregular in unfixed, unstained samples visualized by darkfield microscopy. Image reconstruction studies on fixed and stained ring-shaped specimens indicates that there are 6–10 globular protein elements or patches, each about 3.9 ± 0.5 nm in diameter, per DNA moiety.     

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.     

Expression of the mitochondrial genome in HeLa cells : XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA

HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.     

Euler buckling and nonlinear kinking of double-stranded DNA

The bending stiffness of double-stranded DNA (dsDNA) at high curvatures is fundamental to its biological activity, yet this regime has been difficult to probe experimentally, and literature results have not been consistent. We created a ‘molecular vise’ in which base-pairing interactions generated a compressive force on sub-persistence length segments of dsDNA. Short dsDNA strands (<41 base pairs) resisted this force and remained straight; longer strands became bent, a phenomenon called ‘Euler buckling’. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any ‘kinked’ states. Greater concentrations of monovalent salts or 1 mM Mg²⁺ induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.     

The flexibility of locally melted DNA

Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23°C to 42°C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.     

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with ³H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying ³H-thymidine (10^–6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10^–8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10^–6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.