Crystal structure of 15-mer DNA duplex containing unpaired bases

Errors during DNA replication or repair can lead to the presence of unpaired or inserted bases in the double helix, as well as to mismatched base pairs. So far only structures of the latter type have been characterized by X-ray crystallography. We report here a 3-A crystal structure of DNA 15-mer d(CGCGAAATTTACGCG), which forms a duplex with two unpaired adenine residues looped outside the B-type helix. This arrangement is in disagreement with the nuclear magnetic resonance spectroscopy results for the same 15-mer in solution, indicating polymorphic nature of the structure adopted by this sequence.     

An unusual DNA structure detected in a telomeric sequence under superhelical stress and at low pH

Telomeric sequences of DNA, which are found at the ends of linear chromosomes, have been attracting attention as potential sites for the formation of unusual DNA structures. They consist of (GnTm) or (GnATm) motifs (n greater than or equal to m) and, in the single-stranded state, form hairpins stabilized by non-canonical G.G pairs. In the duplex state and under superhelical stress they exhibit hypersensitivity to SI nuclease which by analogy with homopurine-homopyrimidine sequences may reflect the formation of an unusual structure. To determine whether this is the case we have inserted into a plasmid the Tetrahymena telomeric motif (G4T2).(A2C4) and probed it by two-dimensional gel electrophoresis, chemical modification and oligonucleotide binding. Our data demonstrate that, under superhelical stress and at low pH, the insert does indeed adopt a novel DNA conformation. We have concluded that in this structure the C-rich strand forms a hairpin stabilized by non-Watson-Crick base pairs C.C+ and A.A+, whereas the G-rich strand remains unstructured. We term this new DNA structure the (C,A)-hairpin.     

Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.     

Local destabilisation of a DNA double helix by a T--T wobble pair.

Nuclear magnetic resonance is a technique which permits direct observation of the Waton--Click hydrogen-bonded ring imino protons (guanine N1H and thymine N3H). As the formation and disruption of hydrogen bonds of double-helical RNA and DNA structures are key events during various biological processes, NMR thus provides a useful tool for studying the fluctuational mobility of the individual base pairs. Indeed, several NMR studies of oligo- and polynucleotides have been carried out to probe the structure and dynamics of nucleic acids in solution (for a review see ref. 1). The present study constitutes the first part of our attempt to assess the influence of non-complementary base pairs on the stability of nucleic acid double helices. We report the spectral assignment and temperature-dependent NMR profiles of the hydrogen-bonded imino protons of the two DNA fragments shown in Fig. 1. The assignment is based solely on experimental grounds using the principle of chemical modification. It will be demonstrated that the introduction of a non-complementary (wobble) base pair in a DNA duplex introduces an extra melting site in addition to the sequential melting which starts with the terminal base pairs in the double helix structure.     

Crystal structure of T4 endonuclease VII resolving a Holliday junction

Holliday proposed a four-way DNA junction as an intermediate in homologous recombination, and such Holliday junctions have since been identified as a central component in DNA recombination and repair. Phage T4 endonuclease VII (endo VII) was the first enzyme shown to resolve Holliday junctions into duplex DNAs by introducing symmetrical nicks in equivalent strands. Several Holliday junction resolvases have since been characterized, but an atomic structure of a resolvase complex with a Holliday junction remained elusive. Here we report the crystal structure of an inactive T4 endo VII(N62D) complexed with an immobile four-way junction with alternating arm lengths of 10 and 14 base pairs. The junction is a hybrid of the conventional square-planar and stacked-X conformation. Endo VII protrudes into the junction point from the minor groove side, opening it to a 14 A x 32 A parallelogram. This interaction interrupts the coaxial stacking, yet every base pair surrounding the junction remains intact. Additional interactions involve the positively charged protein and DNA phosphate backbones. Each scissile phosphate that is two base pairs from the crossover interacts with a Mg2+ ion in the active site. The similar overall shape and surface charge potential of the Holliday junction resolvases endo VII, RuvC, Ydc2, Hjc and RecU, despite having different folds, active site composition and DNA sequence preference, suggest a conserved binding mode for Holliday junctions.     

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.     

Structure of an adenine-cytosine base pair in DNA and its implications for mismatch repair

Mutational pathways rely on introducing changes in the DNA double helix. This may be achieved by the incorporation of a noncomplementary base on replication or during genetic recombination, leading to substitution mutation. In vivo studies have shown that most combinations of base-pair mismatches can be accommodated in the DNA double helix, albeit with varying efficiencies. Fidelity of replication requires the recognition and excision of mismatched bases by proofreading enzymes and post-replicative mismatch repair systems. Rates of excision vary with the type of mismatch and there is some evidence that these are influenced by the nature of the neighbouring sequences. However, there is little experimental information about the molecular structure of mismatches and their effect on the DNA double helix. We have recently determined the crystal structures of several DNA fragments with guanine X thymine and adenine X guanine mismatches in a full turn of a B-DNA helix and now report the nature of the base pairing between adenine and cytosine in an isomorphous fragment. The base pair found in the present study is novel and we believe has not previously been demonstrated. Our results suggest that the enzymatic recognition of mismatches is likely to occur at the level of the base pairs and that the efficiency of repair can be correlated with structural features.     

Replication by human DNA polymerase-iota occurs by Hoogsteen base-pairing

Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addition, the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, is an exception to these rules. hPoliota incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here we present the crystal structure of hPoliota bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is 'specialized' for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPoliota opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication.     

X-ray structure of a DNA hairpin molecule

We have solved the crystal structure of a synthetic DNA hexadecanucleotide of sequence: C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G, at 2.1 A resolution, and observed that it adopts a monomeric hairpin configuration with a Z-DNA hexamer stem. In the T4 loop the bases stack with one another and with neighbouring molecules of the crystal, and not with base pairs of their own hexamer stem. Two thymine T10 rings from different molecules stack between the C1-G16 ends of a third and a fourth hairpin helix, in a manner that suggests T-T base 'pairing' and simulates a long, 13-base-pair helix. Although such T-T interactions would not be present in solution, they illustrate a remarkable tendency of thymines for self-association. Purine-purine G-A base pairs are known to exist in the anti-anti conformation with an increase in local helix width; it may be that more serious consideration should be given to the possible existence of pyrimidine-pyrimidine C-T base pairs with decreased local helix width, particularly where several such base pairs occur sequentially.     

Unusual helical packing in crystals of DNA bearing a mutation hot spot

The target sequence of the restriction enzyme NarI (GGCGCC) is a hot spot for the -2 frameshift mutagenesis (GGCGCC----GGCC) induced by the chemical carcinogens such as N-2-acetyl-aminofluorene. Of the guanine residues, all of which show equal reactivity towards the carcinogen, only binding to the 3'-most proximal guanine within the NarI site is able to trigger the frameshift event. We selected the non-palindromic dodecamer d(ACCGGCGCCACA), whose sequence corresponds to the most mutagenic NarI site in pBR322 DNA; for X-ray structure analysis. Its molecular structure determined at 2.8 A resolution reveals significant deviations from the structure of canonical B-form DNA, with partial opening of three G-C base pairs, high propeller twist values and sequence-dependent three-centred hydrogen bonds. This crystal structure shows a novel kind of packing in which helices are locked together by groove-backbone interactions. The partial opening of G-C base pairs is induced by interactions of phosphate anionic oxygen atoms with the amino group of cytosine bases. This provides a model for close approach of DNA molecules during biological processes, such as recombination.     

Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair

The Ku heterodimer (Ku70 and Ku80 subunits) contributes to genomic integrity through its ability to bind DNA double-strand breaks and facilitate repair by the non-homologous end-joining pathway. The crystal structure of the human Ku heterodimer was determined both alone and bound to a 55-nucleotide DNA element at 2.7 and 2.5 A resolution, respectively. Ku70 and Ku80 share a common topology and form a dyad-symmetrical molecule with a preformed ring that encircles duplex DNA. The binding site can cradle two full turns of DNA while encircling only the central 3-4 base pairs (bp). Ku makes no contacts with DNA bases and few with the sugar-phosphate backbone, but it fits sterically to major and minor groove contours so as to position the DNA helix in a defined path through the protein ring. These features seem well designed to structurally support broken DNA ends and to bring the DNA helix into phase across the junction during end processing and ligation.     

Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA

DNA mismatch repair is critical for increasing replication fidelity in organisms ranging from bacteria to humans. MutS protein, a member of the ABC ATPase superfamily, recognizes mispaired and unpaired bases in duplex DNA and initiates mismatch repair. Mutations in human MutS genes cause a predisposition to hereditary nonpolyposis colorectal cancer as well as sporadic tumours. Here we report the crystal structures of a MutS protein and a complex of MutS with a heteroduplex DNA containing an unpaired base. The structures reveal the general architecture of members of the MutS family, an induced-fit mechanism of recognition between four domains of a MutS dimer and a heteroduplex kinked at the mismatch, a composite ATPase active site composed of residues from both MutS subunits, and a transmitter region connecting the mismatch-binding and ATPase domains. The crystal structures also provide a molecular framework for understanding hereditary nonpolyposis colorectal cancer mutations and for postulating testable roles of MutS.     

An unprecedented nucleic acid capture mechanism for excision of DNA damage

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.     

Use of an 125I-labelled DNA ligand to probe DNA structure

It no longer seems likely that DNA molecules in situ have a uniform conformation, represented by the classical B-form helix. For example, recent structural studies have shown that in certain conditions DNA can have a left-handed (so-called Z-form) helix, and have revealed extensive sequence-dependent variations of B-DNA helical parameters. Such sequence-dependent variations in DNA structure can be investigated in solution with reagents that bind to DNA in a conformation-dependent manner, and cut one or both strands of the double-helix at the site of binding, as, for example, has been shown for the endonuclease DNase I3. We describe here a simple way to endow a DNA-binding ligand with the ability to cleave DNA--labelling with 125I. The radiochemical damage associated with 125I decay induces a double-stranded DNA break. Using this technique we have shown that a sequence of four consecutive A X T base pairs is a necessary, but not sufficient, condition for strong binding to DNA of the bis-benzamide Hoechst 33258--presumably the other important factor is the conformation of the double-helix at the site of the (A/T)4 sequence. We suggest 125I-Hoechst 33258 may be a useful new probe of DNA structure.     

Structure of the repressor-operator complex of bacteriophage 434

The crystal structure of a specific complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator DNA shows interactions that determine sequence-dependent affinity. The repressor recognizes its operators by its complementarity to a particular DNA conformation as well as by direct interaction with base pairs in the major groove.     

Atomic-scale imaging of DNA using scanning tunnelling microscopy

The scanning tunnelling microscope (STM) has been used to visualize DNA under water, under oil and in air. Images of single-stranded DNA have shown that submolecular resolution is possible. Here we describe atomic-resolution imaging of duplex DNA. Topographic STM images of uncoated duplex DNA on a graphite substrate obtained in ultra-high vacuum are presented that show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles show excellent correlation with atomic contours of the van der Waals surface of A-form DNA derived from X-ray crystallography. A comparison of variations in the barrier to quantum mechanical tunnelling (barrier-height) with atomic-scale topography shows correlation over the phosphate-sugar backbone but anticorrelation over the base pairs. This relationship may be due to the different chemical characteristics of parts of the molecule. Further investigation of this phenomenon should lead to a better understanding of the physics of imaging adsorbates with the STM and may prove useful in sequencing DNA. The improved resolution compared with previously published STM images of DNA may be attributable to ultra-high vacuum, high data-pixel density, slow scan rate, a fortuitously clean and sharp tip and/or a relatively dilute and extremely clean sample solution. This work demonstrates the potential of the STM for characterization of large biomolecular structures, but additional development will be required to make such high resolution imaging of DNA and other large molecules routine.     

Random components in mutagenesis

The mutability of DNA varies enormously from one base pair to another. Part of this variation is due to the specificity of the reaction between mutagens and base, but much of the variation is due to unknown causes. A genetic system developed by Miller and colleagues allows the mutation frequencies of a large number of different base pairs in the lacI gene of Escherichia coli to be compared. For example, Coulondre and Miller found that the sites most readily mutated by UV light are almost 100 times more often mutated than the least susceptible sites. A recently completed study of mutagenesis with neocarzinostatin (NCS) in the lacI gene has prompted us to re-examine some previous studies, of mutagenesis in this gene. Our analysis, reported here, suggests that the mutations induced by certain mutagens fall into two classes: mutations in one class are clearly distributed non-randomly, that is, they are very common at some sites and significantly less common at others; mutations in the second class, however, occur at low frequency and appear to be randomly distributed. Both classes of mutations seem to occur only at damaged bases.