RNA-DNA hybrids containing damaged DNA are substrates for RNase H.

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.

Magnesium increases the curvature of duplex DNA that contains dA tracts.

Distinct structural features of DNA, such as the curvature of dA tracts, are important in the recognition, packaging, and regulation of DNA. Physiologically relevant concentrations of magnesium have been found to enhance the curvature of dA tract DNAs, as monitored by solution-state NMR, indicating that the structure of DNA depends on the cations present in solution. A model is presented which accounts for the sequence-dependent effects of magnesium on DNA curvature as well as for the previously known sequence-independent effect on DNA flexibility.

Functional and dysfunctional roles of quadruplex DNA in cells.

A number of biological roles have been proposed for quadruplex, also referred to as G4 or tetraplex, DNA. The presence of quadruplex DNA may lead to errors in some biological processes and be required in others. Proteins that interact with quadruplex DNA have been identified including those that cause Bloom's and Werner's syndromes. There are small molecules that specifically bind to quadruplex DNA, inhibit telomerase, and are cytotoxic towards tumor cells indicating a role for quadruplex DNA in telomere function. It is now possible to make testable proposals for the possible biological implications of quadruplex DNA in replication, transcription, and recombination as well as possible routes to therapeutic intervention.

The curvature of dA tracts is temperature dependent.

The curvature of dA tracts has been proposed to be important in the recognition, packaging, and regulation of DNA. The effects of dA tracts on the gel mobility, rate of cyclization, and other properties of DNA have been extensively studied. The consensus value for the curvature induced by a single dA tract is about 18 degrees. There are two main competing models for the origin of the curvature of dA tracts. One model assigns the central role to sequence-dependent steric clashes and the other to sequence dependent interactions with cations. The temperature dependence of the shape functions, the molecule specific part of the diffusion coefficients, of a set of six DNAs has been examined here. The set contains DNAs with dA tracts in or out of phase with respect to the helical repeat as well as those with scrambled dA-dT regions. The results show that the curvature of dA tracts is highly temperature dependent and that the curvature is largely melted out by 40 degrees C. The curvature melts out before there is significant premelting, or breathing of the dA tracts or the scrambled dA-dT regions. The curvature does not appear to reach a plateau value at low temperatures. A qualitative model for the melting of the curvature of dA tracts is proposed.

Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA.

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium-quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium-DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.

Flexibility and curvature of duplex DNA containing mismatched sites as a function of temperature.

The flexibility and curvature of duplex DNAs containing mismatched sites have been monitored as a function of temperature. The diffusion coefficients are dependent on the flexibility and the curvature of the DNA, and these have been determined by NMR-based methods. The diffusion coefficients, D, depend on a Boltzmann term and the viscosity of the solvent, eta, which is also temperature dependent. To analyze the temperature dependence of the diffusion results, the shape function, S(f) = etaD/T, is used. The shape functions do not have the viscosity and temperature dependence of the diffusion coefficients. The presence of mismatched sites significantly enhances the shape function of duplex DNA at all temperatures examined. The observed increases in the shape functions are attributed to the mismatched sites acting as localized flexible joints. The results on the temperature dependence of the shape functions, the optical absorbance, and the proton chemical shifts indicate that local melting at, and adjacent to, mismatched site occurs at a lower temperature than the overall melting of the duplexes. The localized melting gives rise to a considerable increase in the shape function. The contribution of the curvature of the mismatched sites to the enhanced diffusion has been examined. A DNA with mismatches that are in phase with respect to the helical repeat and a DNA which has the mismatches out of phase with respect to the helical repeat have been examined. The results indicate that mismatched sites have modest curvature.

6-Thioguanine alters the structure and stability of duplex DNA and inhibits quadruplex DNA formation.

The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.

Determinants of DNA quadruplex structural type: sequence and potassium binding.

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.

Damage increases the flexibility of duplex DNA.

It is proposed that much of the recognition of specific types of damaged DNAs is based on accessible structural features, while much of the recognition of damaged DNAs, as a class, is based on flexibility. The more flexible a DNA the faster its diffusion rate. The diffusion rates of each member of a series of damaged duplex DNAs has been found to be significantly faster than that of the corresponding undamaged duplex DNA. The damaged sites studied include apurinic and apyrimidinic a basic sites, thymine glycol and urea. The presence of mismatched sites also increases the diffusion. Thus, damaged DNAs appear to have sufficient flexibility for recognition and the flexibility may allow damaged sites to act as a universal joint or hinge that allows distant sites on the DNA to come together.

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Porphyrins can catalyze the interconversion of DNA quadruplex structural types.

The binding of porphyrins to quadruplex DNAs provides a model system for the examination of drug binding to telomere, centromere, triplet repeat and other DNAs which may form quadruplex structures in vivo. Porphyrins, and certain other molecules that interact with quadruplex DNAs, have been shown to have significant biological activity. In this investigation the interactions of porphyrins with quadruplex DNAs have been examined by optical and NMR methods. The fluorescence of selected porphyrins can be used to discriminate between duplex and quadruplex DNAs. The fluorescence of the porphyrins can also be used to discriminate partially between the chair, basket and parallel stranded types of quadruplex DNA. At the relatively high DNA concentrations used in NMR, the porphyrins catalyze the conversion of both chair and basket type structures into parallel strand quadruplex DNAs. A DNA-porphyrin system has been found which appears to be a model for an intermediate of the catalytic pathway.

Determination of internuclear angles of DNA using paramagnetic-assisted magnetic alignment.

Paramagnetic ions have been used to assist the magnetic alignment of DNA. The anisotropy of the binding sites is sufficient to give rise to significant alignment of the DNA with the observed proton-carbon dipolar couplings spanning a 70-Hz range. The dipolar couplings have been used to determine the positions of the axial and rhombic alignment axes. The positions of the alignment axes relative to the positions of the binding sites of the paramagnetic europium ions have also been determined.

Fluorescent dyes specific for quadruplex DNA.

Fluorescent dyes which are specific for duplex DNA have found a wide range of applications from staining gels to visualization of chromosomes. Porphyrin dyes have been found which are highly fluorescent in the presence of quadruplex but not duplex DNA. These dyes may offer a route to the specific detection of quadruplex DNA under biologically important conditions. There are three types of DNA quadruplex structures, and these may play important roles in telomere, centromere, triplet repeat, integration sites and other DNAs, and this first set of porphyrin dyes show some selectivity between the quadruplex types.

Interresidue quiet NOEs for DNA structural studies.

The potential utility of long-range NOEs in DNA has not been exploited since the observed signals have contributions both from the direct magnetization route and from multiple diffusion pathways. The Quiet NOE approach can be used to select for the direct magnetization transfer pathway by suppressing spin diffusion. A single-band Quiet NOE, which allows detection of the direct NOEs between protons in a selected chemical shift window, has been demonstrated on two duplex DNAs, and the NOEs observed can contain important structural information.

Replication inhibition and miscoding properties of DNA templates containing a site-specific cis-thymine glycol or urea residue.

Oligodeoxynucleotides modified site-specifically with cis-thymine glycol or urea residue, two ionizing radiation/oxidation damages, were used as templates in primer extension reactions catalyzed by 3' --> 5' exonuclease-deficient Klenow fragment, human DNA polymerase beta, AMV reverse transcriptase, and a modified T7 DNA polymerase (Sequenase). Both lesions blocked DNA replication one nucleotide before and opposite the lesion site, but a significant fraction of full-length product was obtained after prolonged incubation. Hill plot analysis of the results on both thymine glycol- and urea- containing templates by 3' --> 5' exonuclease-deficient Klenow fragment for incorporation of either dATP or dGTP gave linear plots with Hill coefficients much less than 1. This suggests that the dNTP concentration influences the termination of DNA synthesis at multiple steps of the catalytic process. The specificity of nucleotide incorporation opposite these lesions and chain extension by the same polymerase was determined by a steady-state kinetic analysis. The kinetic studies established that the rate of nucleotide incorporation and chain extension was highest with deoxyadenosine opposite both these lesions. However, the efficiency of forming a G.T pair relative to an A.T pair for the control at a level of 1/10(9) was enhanced to approximately 1/160 for thymine glycol and 1/20 for urea, although the former lesion was more bypassable than the latter lesion. On the basis of these in vitro results, we conclude that both these DNA damages are impediments of DNA synthesis and that a urea residue, in particular, has the potential to miscode.

Structures of apurinic and apyrimidinic sites in duplex DNAs.

Natural and exogenous processes can give rise to abasic sites with either a purine or pyrimidine as the base on the opposing strand. The solution state structures of the apyrimidinic DNA duplex, with D6 indicating an abasic site, [sequence: see text] referred to as AD, and the apurinic DNA duplex with a dC17, referred to as CD, have been determined. A particularly striking difference is that the abasic site in CD is predominantly a beta hemiacetal, whereas in AD the alpha and beta forms are equally present. Hydrogen bonding with water by the abasic site and the base on the opposite strand appears to play a large role in determining the structure near the damaged site. Comparison of these structures with that of a duplex DNA containing a thymine glycol at the same position as the abasic site and with that of a duplex DNA containing an abasic site in the middle of a curved DNA sequence offers some insight into the common and distinct structural features of damaged DNA sites.

Purification of thymine glycol DNA and nucleosides by use of boronate chromatography.

Boronate columns can be used to purify DNAs containing cis-thymine glycol residues and can also be used to purify cis-thymine glycol nucleosides. The boronate group can form a reversible complex with the cis-diol of the thymine glycol but not with the urea residue which is formed by alkaline hydrolysis of the thymine glycol. This method is rapid and appears applicable to a range of nucleic acids. In addition to the purifications of DNAs and nucleosides demonstrated here boronate chromatography may be applicable to assaying the extent and sites of oxidative damage to DNAs.

Protein phi and psi dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures.

The chemical shifts of the backbone atoms of proteins can be used to obtain restraints that can be incorporated into structure determination methods. Each chemical shift can be used to define a restraint and these restraints can be simultaneously used to define the local, secondary structure features. The global fold can be determined by a combined use of the chemical shift based restraints along with the long-range information present in the NOEs of partially deuterated proteins or the amide-amide NOEs but not from such limited NOE data sets alone. This approach has been demonstrated to be capable of determining the overall folding pattern of four proteins. This suggests that solution-state NMR methods can be extended to the structure determination of larger proteins by using the information present in the chemical shifts of the backbone atoms along with the data that can be obtained on a small number of labeled forms.

Solution structure of a duplex DNA with an abasic site in a dA tract.

The presence of dA tracts in DNA can lead to stable curvature of the DNA, and this curvature can be important in gene regulation, DNA packaging, and other processes. Since damage to DNA may eliminate this stable curvature, the solution state structure of the duplex of d(CGCAAAAATGCG) paired with d(CGCATTDTTCCG), with D indicating an abasic site, has been determined. The undamaged DNA bends into the major groove both in solution and in the crystal state. The presence of the abasic site in the dA tract region induces changes in the DNA structure up to four base pairs away from the damaged site. The structure of the DNA is dependent on whether the abasic site is in the alpha or beta hemiacetal form. These consequences are quite different from the more localized effects that have been observed for "normal" DNAs containing abasic sites. Thus, there appears to be a strong sequence dependence of the structural effects of abasic sites just as there is for undamaged DNA. Furthermore, these results indicate that the presence of an abasic site can alter DNA bending and hence is likely to have significant long range effects on gene regulation and other properties that are dependent on the stable curvature of DNA.

Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase.

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung.Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung.Ugi association and irreversible complex formation.