Footprinting studies of DNA-sequence recognition by nogalamycin.

We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase-I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand-binding sites surrounded by repetitive sequences such as (A)n.(T)n and (AT)n. Within regions of (A)n.(T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 microM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 microM), nogalamycin prevents DNAse-I cleavage of (AT)n but shows no interaction with regions of (AC)n.(GT)n. Regions of (A)n.(T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.     

P1 nuclease cleavage is dependent on length of the mismatches in DNA

P1 nuclease is one of the most extensively used single-strand DNA specific nucleases in molecular biology. In modern biology, it is used as an enzymatic probe to detect altered DNA conformations. It is well documented that P1 cleaves single-stranded nucleic acids and single-stranded DNA regions. The fact that P1 can act under a wide range of conditions, including physiological pH and temperature make it the most commonly used enzymatic probe in DNA structural studies. Surprisingly, to this date, there is no study to characterize the influence of length of mismatches on P1 sensitivity. Using a series of radioactively labeled oligomeric DNA substrates-containing mismatches, we find that P1 nuclease cleavage is dependent on the length of mismatches. P1 does not cleave DNA when there is a single-base mismatch. P1 cleavage efficiency is optimum when mismatch length is 3 nt or more. Changing the position of the mismatches also does not make any difference in cleavage efficacy. These novel findings on P1 properties have implications for its use in DNA structure and DNA repair studies.     

Finding and Characterizing the Complexes of Drug Like Molecules with Quadruplex DNA: Combined Use of an Enhanced Hydroxyl Radical Cleavage Protocol and NMR

Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the chair type quadruplex formed by the thrombin binding aptamer, d(GGTTGGTGTGGTTGG). The hydroxyl radical results indicate that NSC 176319, Cain’s quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes.     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Energetics of DNA end binding by E.coli RecBC and RecBCD helicases indicate loop formation in the 3'-single-stranded DNA tail

We examined the equilibrium binding of Escherichia coli RecBC and RecBCD helicases to duplex DNA ends possessing pre-existing single-stranded (ss) DNA ((dT)(n)) tails varying in length (n=0 to 20 nucleotides) in order to determine the contributions of both the 3' and 5' single strands to the energetics of complex formation. Protein binding was monitored by the fluorescence enhancement of a reference DNA labeled at its end with a Cy3 fluorophore. Binding to unlabeled DNA was examined by competition titrations with the Cy3-labeled reference DNA. The affinities of both RecBC and RecBCD increase as the 3'-(dT)(n) tail length increases from zero to six nucleotides, but then decrease dramatically as the 3'-(dT)(n) tail length increases from six to 20 nucleotides. Isothermal titration calorimetry experiments with RecBC show that the binding enthalpy is negative and increases in magnitude with increasing 3'-(dT)(n) tail length up to n=6 nucleotides, but remains constant for n > or =6. Hence, the decrease in binding affinity for 3'-(dT)(n) tail lengths with n > or =6 is due to an unfavorable entropic contribution. RecBC binds optimally to duplex DNA with (dT)6 tails on both the 3' and 5'-ends while RecBCD prefers duplex DNA with 3'-(dT)6 and 5'-(dT)10 tails. These data suggest that both RecBC and RecBCD helicases can destabilize or "melt out" six base-pairs upon binding to a blunt DNA duplex end in the absence of ATP. These results also provide the first evidence that a loop in the 3'-ssDNA tail can form upon binding of RecBC or RecBCD with DNA duplexes containing a pre-formed 3'-ssDNA tail with n > or =6 nucleotides. Such loops may be representative of those hypothesized to form upon interaction of a Chi site contained within the unwound 3' ss-DNA tail with the RecC subunit during DNA unwinding.     

Oligo(dT) is not a correct native PAGE marker for single-stranded DNA

Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility, and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length, and gel concentration. Depending on their length, oligo(dT) fragments migrate 2--4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g., determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.     

Hydration and conformational transitions in DNA, RNA, and mixed DNA-RNA triplexes studied by gravimetry and FTIR spectroscopy

We have studied by gravimetric measurements and FTIR spectroscopy the hydration of duplexes and triplexes formed by combinations of dA(n), dT(n), rA(n), and rU(n) strands. Results obtained on hydrated films show important differences in their hydration and in the structural transitions which can be induced by varying the water content of the samples. The number of water molecules per nucleotide (w/n) measured at high relative humidity (98% R.H.) is found to be 21 for dA(n).dT(n) and 15 for rA(n).rU(n). Addition of a third rU(n) strand does not change the number of water molecules per nucleotide: w/n=21 for rU(n)*dA(n).dT(n) and w/n=15 for rU(n)*rA(n).rU(n). On the contrary, the addition of a third dT(n) strand changes the water content but in a different way, depending whether the duplex is DNA or RNA. Thus, a loss of four water molecules per nucleotide is measured for dT(n)*dA(n).dT(n) while an increase of two water molecules per nucleotide is observed for dT(n)*rA(n).rU(n). The final hydration is the same for both triplexes (w/n=17). The desorption profiles obtained by gravimetry and FTIR spectroscopy are similar for the rA(n).rU(n) duplex and the rU(n)*rA(n).rU(n) triplex. On the contrary, the desorption profiles of the dA(n).dT(n) duplex and the triplexes formed with it (rU(n)*dA(n).dT(n) and dT(n)*dA(n).dT(n)) are different from each other. This is correlated with conformational transitions induced by varying the hydration content of the different structures, as shown by FTIR spectroscopy. Modifications of the phosphate group hydration and of the sugar conformation (S to N type repuckering) induced by decrease of the water content are observed in the case of triplexes formed on the dA(n).dT(n) duplex.     

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair(NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired.     

The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles).

We have studied the structure and reactivities of two kinds of mismatched DNA sequences--unopposed bases, or bulges, and multiple mismatched pairs of bases. These were generated in a constant sequence environment, in relatively long DNA fragments, using a technique based on heteroduplex formation between sequences cloned into single-stranded M13 phage. The mismatched sequences were studied from two points of view, viz 1. The mobility of the fragments on gel electrophoresis in polyacrylamide was studied in order to examine possible bending of the DNA due to the presence of the mismatch defect. Such bending would constitute a global effect on the conformation of the molecule. 2. Sequences in and around the mismatches were studied using enzyme and chemical probes of DNA structure. This would reveal more local structural effects of the mismatched sequences. We observed that the structures of the bulges and the multiple mismatches appear to be fundamentally different. The bulged sequences exhibited a large gel retardation, consistent with a significant bending of the DNA at the bulge, and whose magnitude depends on the number of mismatched bases. The larger bulges were sensitive to cleavage by single-strand specific nucleases, and modified by diethyl pyrocarbonate (adenines) or osmium tetroxide (thymines) in a non-uniform way, suggesting that the bulges have a precise structure that leads to exposure of some, but not all, of the bases. In contrast the multiple mismatches ('bubbles') cause very much less bending of the DNA fragment in which they occur, and uniform patterns of chemical reactivity along the length of the mismatched sequences, suggesting a less well defined, and possibly flexible, structure. The precise structure of the bulges suggests that such features may be especially significant for recognition by proteins.     

DNA mismatch repair: the hands of a genome guardian

DNA mismatch repair (MMR) is initiated when the MutS protein recognizes damaged DNA. Crystal structures of MutS bound to mispaired and unpaired DNA show how MutS distinguishes damaged from undamaged DNA and explain how a broad variety of DNA mismatch lesions can be detected. The structures suggest mechanisms for the ATP-induced structural regulation of multistep DNA repair processes.     

DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands

DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase–polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase–polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.     

The elongation of mismatched primers by DNA polymerase alpha from calf thymus

The ability of the 9S and 5.7S DNA polymerase alpha subspecies from calf thymus in elongating a mismatched primer terminus has been investigated. With poly(dA) as template, the elongation rate for (dT)8dG, (dT)8dC and (dT)10dGdT is 20-fold lower for the 9S enzyme and 5-fold lower for the 5.7S enzyme as compared to (dT)10. The presence of a second mismatch at the primer terminus reduces the elongation rate further by a factor of two. Exonucleolytic excision of the mismatches can be excluded. With (dT)8dG (dT)n as primer we show, that at least five T-residues have to follow the mismatch in order to establish the elongation rate of a perfectly paired primer. The KM value for (dT)10 dG as primer is 400 nM as compared to 10 nM for (dT)10. Addition of Mn2+ increases the relative efficiency of elongation of the mismatched primers.     

Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS alpha and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS alpha for the mismatch. In contrast, the damaged bases in these compound lesions were excised three- to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS alpha in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.     

Effects of the antitumor antibiotic mithramycin on the structure of repetitive DNA regions adjacent to its GC-rich binding site.

Regions of An.Tn, (GA)n.(TC)n, and (GT)n.(AC)n have been cloned into the SmaI (CCC/GGG) site of plasmid pUC19. HindIII-EcoRI restriction fragments containing these inserts have been used as substrates for footprinting experiments using DNase I, DNase II, and micrococcal nuclease as probes. These present good mithramycin binding sites (GGG) flanking repetitive regions to which the drug does not bind. In each case, mithramycin footprints are observed at the CCC/GGG sites, which are not affected by the nature of the surrounding sequences. Some weaker binding is detected at TCGA and ACCA sites and at regions of alternating GA. No binding is found to regions of alternating GT. An.Tn inserts (n = 23 or 69) are normally resistant to cleavage by all these probes; in the presence of mithramycin, a dramatic increase in DNase I cleavage is observed throughout the entire insert and is indicative of an alteration in DNA structure. Similar changes are seen with DNase II and micrococcal nuclease. These changes cannot be explained by invoking changes in the ratio of free substrate to cleavage agent. In contrast, cleavage of (GA)n.(CT)n and (GT)n.(AC)n inserts is not affected by drug binding. The results are consistent with a model in which mithramycin causes dramatic changes in the width of the DNA minor groove, generating a structure which has some properties of A-DNA, and suggest that this can be propagated into surrounding DNA regions in a sequence-dependent manner. The structural alterations with An.Tn are highly cooperative and can be transmitted over at least three turns of the DNA helix.     

Molecular mechanisms of DNA mismatch repair.

DNA mismatch repair (MMR) safeguards the integrity of the genome. In its role in postreplicative repair, this repair pathway corrects base-base and insertion/deletion (I/D) mismatches that have escaped the proofreading function of replicative polymerases. In its absence, cells assume a mutator phenotype in which the rate of spontaneous mutation is greatly elevated. The discovery that defects in mismatch repair segregate with certain cancer predisposition syndromes highlights its essential role in mutation avoidance. Recently, three-dimensional structures of MutS, a key repair protein that recognizes mismatches, have been determined by X-ray crystallography. This article provides an overview of the structural features of MutS proteins and discusses how the structural data together with biochemical and genetic studies reveal new insights into the molecular mechanisms of mismatch repair.     

DNA mismatch repair: MutS structures bound to mismatches

When DNA mismatch repair fails, the result is a mutator phenotype, which can lead to cancer in humans. Functional repair is dependent on the recognition of mismatches by a dimeric MutS protein, a homodimer in bacteria but a heterodimer in humans. Recent crystal structures of Thermus aquaticus and Escherichia coli MutS have revealed the structural heterodimeric nature of the bacterial proteins and provide new insights into their complicated ATP-dependent repair mechanism.