Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Multiplexed DNA-mediated electrochemistry

The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of π-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the π-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA π-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.

First, Methylene Blue (MB′) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB′ was reduced through DNA-mediated charge transport. The redox signal intensity for MB′-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB′ on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.

Next, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening π-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA. Further gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.

Finally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.

Investigations of DNA-mediated protein oxidation

DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]^3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.

Further work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)₂, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)₂ is substituted with poly(dAdT)₂, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.

We have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.

DNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

While the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]^3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]³⁺ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.

In conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Studies on the role of histones in the structure and function of chromatin

Studies on the dissociation of histones from chromatin by increasing concentrations of sodium deoxycholate (DOC) have shown that histrone II is removed at lowest concentrations of DOC, while slightly higher concentrations remove histones III and IV. Still higher concentrations remove histone I.

The complete separation of chromatin and ¹⁴C-DOC by sucrose sedimentation indicated that the binding of DOC to chromatin is readily and completely reversible.

The dissociation of histones from chromatin by increasing concentrations of related cholanic acids and some of their conjugated derivatives were studied. The results suggested that the driving force for the interaction between the cholanic acid anion and histones is the lowering of the activity coefficient of the cholanic acid anion which occurs when it is partially removed from solution by interaction with hydrophobic regions of the positively charged histones.

The role of histones in the structure of chromatin has been studied by comparing the effects of selective removal of histones from chromatin by increasing concentrations of DOC with those caused by NaCl (removes histone I at lowest concentrations, while higher concentrations remove histones II, III, and IV). Properties studied included thermal denaturation, sedimentation velocity, flow dichroism, relaxation times of molecules oriented in a flow field, and the irreversible disruption of a 130 S, cross-linked component of sheared chromatin. The data indicated that none of the structural or chemical parameters with which these properties are correlated show a dependence on the presence of one particular histone fraction.

The template activity (ability to prime a 0.2 M KC1 DNA-dependent RNA synthesis system catalyzed by E. coli RNA polymerase) increases from that of native chromatin (approximately 25 per cent of that pure DNA) to that of pure DNA in a fashion which shows a nearly linear relationship to the amount of histone coverage of the template. The precipitability of partially dehistonized chromatin samples in 0.15 M NaCl shows a large dependence on the presence of histone I.

The interactions of basic proteins and DNA

I. ELECTROPHORESIS OF THE NUCLEIC ACIDS

A zone electrophoresis apparatus using ultraviolet optics has been constructed to study nucleic acids at concentrations less than 0.004%. Native DNA has a mobility about 15% higher than denatured DNA over a range of conditions. Otherwise, the electrophoretic mobility is independent of molecular weight, base composition or source. DNA mobilities change in the expected way with pH but the fractional change in mobility is less than the calculated change in charge. A small decrease in mobility accompanies an increase in ionic strength. RNA’s from various sources have mobilities slightly lower than denatured DNA except for s-RNA which travels slightly faster. The important considerations governing the mobility of nucleic acids appear to be the nature of the hydrodynamic segment, and the binding of counterions. The differences between electrophoresis and sedimentation stem from the fact that all random coil polyelectrolytes are fundamentally free draining in electrophoresis.

II. THE CYTOCHROME C/DNA COMPLEX

The basic protein, cytochrome c, has been complexed to DNA. Up to a cytochrome:DNA mass ratio of 2, a single type of complex is formed. Dissociation of this complex occurs between 0.05F and 0.1F NaCl. The complexing of cytochrome to DNA causes a slight increase in the melting temperature of the DNA, and a reduction of the electrophoretic mobility proportional to the decrease in net charge. Above a cytochrome:DNA mass ratio of 2.5, a different type of complex is formed. The results suggest that complexes such as are formed in the Kleinschmidt technique of electron microscopy would not exist in bulk solution and are exclusively film phenomena.

III. STUDIES OF THE ELECTROPHORESIS AND MELTING BEHAVIOUR OF NUCLEOHISTONES

Electrophoresis studies on reconstituted nucleohistones indicate that the electrophoretic mobility for these complexes is a function of the net charge of the complex. The mobility is therefore dependent on the charge density of the histone complexing the DNA, as well as on the histone/DNA ratio. It is found that the different histones affect the transition from native to denatured DNA in different ways. It appears that histone I is exchanging quite rapidly between DNA molecules in 0.01 F salt, while histone II is irreversibly bound. Histone III-IV enhances the capacity of non-strand separated denatured DNA to reanneal. Studies on native nucleoproteins indicate that there are no gene-sized uncomplexed DNA regions in any preparations studied.

IV. THE DISSOCIATION OF HISTONE FROM CALF THYMUS CROMATIN

Calf thymus nucleoprotein was treated with varying concentrations of NaCl. The identity of the histones associated and dissociated from the DNA at each salt concentration was determined by gel electrophoresis. It was found that there is no appreciable histone dissociation below 0.4 F NaCl. The lysine rich histones dissociate between 0.4 and 0.5 F NaCl. Their dissociation is accompanies by a marked increase in the solubility of the chromatin. The moderately lysine rich histones dissociate mainly between 0.8 and 1.1 F NaCl. There are two arginine rich histone components: the first dissociates between 0.8 F and 1.1 F NaCl, but the second class is the very last to be dissociated from the DNA (dissociation beginning at 1.0 F NaCl). By 2.0 F NaCl, essentially all the histones are dissociated.

The properties of the extracted nucleoprotein were studied. The electrophoretic mobility increases and the melting temperature decreases as more histones are dissociated from the DNA. A comparison with the dissociation of histones from DNA in NaClO₄ shows that to dissociate the same class of histones, the concentration of NaCl required is twice that of NaClO₄.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Cleavage of DNA by polyamide-seco-CBI conjugates

Small molecules that bind to any predetermined DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. Polyamides containing N-methylimidazole (Im) N-methylpyrrole (Py) are cell permeable small molecules that bind DNA according to a set of "pairing rules" with affinities and specificities similar to many naturally occurring DNA binding proteins. Py-Im polyamides offer a general approach to the chemical regulation of gene expression. We demonstrate here that polyamide containing a DNA alkylating moiety seco-CBI can specifically direct sequence specific DNA alkylation. We can also control the strand of DNA that is alkylated, depending on the enantiomer of seco-CBI used and the orientation of the polyamide relative to the alkylation site (Chapter 2). This class of molecules has been applied to a gene repair system in collaboration with the Baltimore group at Caltech (Chapter 3). Also reported are additional seco-CBI polyamide conjugates synthesized to study other systems (HIV-1 and COX-2) (Appendix 1).

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Investigations of Pyrrole-Imidazole Polyamide Effects on DNA Replication

Pyrrole–Imidazole polyamides are programmable, cell-permeable small molecules that bind in the minor groove of double-stranded DNA sequence-specifically. Polyamide binding has been shown to alter the local helical structure of DNA, disrupt protein-DNA interactions, and modulate endogenous gene expression. Py–Im polyamides targeted to the androgen receptor-DNA interface have been observed to decrease expression of androgen-regulated genes, upregulate p53, and induce apoptosis in a hormone-sensitive prostate cancer cell line. Here we report that androgen response element (ARE)-targeted polyamides induced DNA replication stress in a hormone-insensitive prostate cancer cell line. The ATR checkpoint kinase was activated in response to this stress, causing phosphorylation of MCM2, and FANCD2 was monoubiquitinated. Surprisingly, little single-stranded DNA was exhibited, and the ATR targets RPA2 and Chk1 were not phosphorylated. We conclude that polyamide induces relatively low level replication stress, and suggest inhibition of the replicative helicase as a putative mechanism based on in vitro assays. We also demonstrate polyamide-induced inhibition of DNA replication in cell free extracts from X. laevis oocytes. In this system, inhibition of chromatin decondensation is observed, preventing DNA replication initiation. Finally, we show that Py-Im polyamides targeted to the ARE and ETS binding sequence downregulate AR- and ERG-driven signaling in a prostate cancer cell line harboring the TMPRSS2-ERG fusion. In a mouse xenograft model, ARE-targeted polyamide treatment reduced growth of the tumor.

Investigations of nonhistone chromosomal proteins

The major nonhistone chromosomal proteins (NHC proteins) are a group of 14-20 acidic proteins associated with DNA in eukaryotic chromatin. In comparisons by SDS gel electrophoresis (molecular weight sieving) one observes a high degree of homology among the NHC protein fractions of different tissues from a given species. Tissue-specific protein bands are also observed. The appearance of a new NHC protein, A, in the NHC proteins of rat liver stimulated to divide by partial hepatectomy and of rat ascites cells suggests that this protein may play a role in preparing the cell for division. The NHC proteins of the same tissue from different species are also very similar. Quantitative but not qualitative changes in the NHC proteins of rat uterus are observed on stimulation (in vivo) with estrogen. These observations suggest that the major NHC proteins play a general role in chromatin structure and the regulation of genome expression; several may be enzymes of nucleic acid and histone metabolism and/or structural proteins analogous to histones. One such enzyme, a protease which readily and preferentially degrades histones, can be extracted from chromatin with 0.7 N NaCl.

Although the NHC proteins readily aggregate, they can be separated from histone and fractionated by ion exchange chromatography on Sephadex SE C-25 resin in 10 M urea-25% formic acid (pH 2.5). Following further purification, four fractions of NHC protein are obtained; two of these are single purified proteins, and the other two contain 4-6 and 4-7 different proteins. These NHC proteins show a ratio of acidic to basic amino acids from 2.7 to 1.2 and isoelectric points from apparently less than 3.7 to 8.0. These isolated fractions appear more soluble and easier to work with than any whole NHC protein preparation.