Molecular structure of a complete turn of A-DNA

We have determined the crystal structure of the dodecamer d(CCCCCGCGGGGG), showing for the first time a complete turn of A-DNA. It has average structural parameters similar to those determined in fibres. Nevertheless it shows a considerable local variation in structure which is in part associated with the presence of a bound spermine molecule. We conclude that the local DNA conformation does not only depend on the base sequence, but may be strongly modified upon interaction with other molecules. In particular, the CpG sequence, which is found in hypersensitive regions of the genome, appears to be able to easily change its conformation under external influences.     

Rotational and translational positions determine the structural and dynamic impact of a single ribonucleotide incorporated in the nucleosome

Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, causing replication stress, chromosome fragility, gross chromosomal rearrangements, and other mutagenic events. Furthermore, nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. Notably, nucleosome formation is significantly reduced by a single ribonucleotide. Single ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme, which incises the DNA backbone on the 5′-side of the ribonucleotide. While the structural implications of a single ribonucleotide in free duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the possible consequent impact on RER, have not been explored. We have carried out 3.5 μs molecular dynamics simulations of a single ribonucleotide incorporated at various translational and rotational positions in a nucleosome core particle. We find that the presence of the 2′−OH group on the ribose impacts the local conformation and dynamics of both the ribonucleotide and nearby DNA nucleotides as well as their interactions with histones; the nature of these disturbances depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. The ribonucleotide’s preferred C3′-endo pucker is stabilized by interactions with the histones, and furthermore the ribonucleotide can cause dynamic local duplex disturbance involving an abnormal C3′-endo population of the adjacent deoxyribose pucker, minor groove opening, ruptured Watson-Crick pairing, and duplex unwinding that are governed by translation-dependent histone-nucleotide interactions. Possible effects of these disturbances on RER are considered.     

The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ

Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2'-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein-nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.     

Ribonucleotide incorporation,proofreading and bypass by human DNA polymerase δ

In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols α, δ and ?). They are subsequently removed by RNase H2-dependent repair, which if defective leads to replication stress and genome instability. To extend these studies to humans, where an RNase H2 defect results in an autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the 3′-exonuclease activity of yeast and human Pol δ largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses ribonucleotides in DNA templates, with four consecutive ribonucleotides in a DNA template being more problematic than single ribonucleotides. In conjunction with recent studies in yeast and mice, this ribonucleotide incorporation may be relevant to impaired development and disease when RNase H2 is defective in mammals. As one tool to investigate ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol δ.     

Influence of counter-ions on the crystal structures of DNA decamers: binding of [Co(NH3)6]3+ and Ba2+ to A-DNA.

A-DNA is a stable alternative right-handed double helix that is favored by certain sequences (e.g., (dG)n.(dC)n) or under low humidity conditions. Earlier A-DNA structures of several DNA oligonucleotides and RNA.DNA chimeras have revealed some conformational variation that may be the result of sequence-dependent effects or crystal packing forces. In this study, four crystal structures of three decamer oligonucleotides, d(ACCGGCCGGT), d(ACCCGCGGGT), and r(GC)d(GTATACGC) in two crystal forms (either the P6(1)22 or the P2(1)2(1)2(1) space group) have been analyzed at high resolution to provide the molecular basis of the structural difference in an experimentally consistent manner. The study reveals that molecules crystallized in the same space group have a more similar A-DNA conformation, whereas the same molecule crystallized in different space groups has different (local) conformations. This suggests that even though the local structure is influenced by the crystal packing environments, the DNA molecule adjusts to adopt an overall conformation close to canonical A-DNA. For example, the six independent CpG steps in these four structures have different base-base stacking patterns, with their helical twist angles (omega) ranging from 28 degrees to 37 degrees. Our study further reveals the structural impact of different counter-ions on the A-DNA conformers. [Co(NH3)6]3+ has three unique A-DNA binding modes. One binds at the major groove side of a GpG step at the O6/N7 sites of guanine bases via hydrogen bonds. The other two modes involve the binding of ions to phosphates, either bridging across the narrow major groove or binding between two intra-strand adjacent phosphates. Those interactions may explain the recent spectroscopic and NMR observations that [Co(NH3)6]3+ is effective in inducing the B- to A-DNA transition for DNA with (G)n sequence. Interestingly, Ba2+ binds to the same O6/N7 sites on guanine by direct coordinations.     

Neomycin, spermine and hexaamminecobalt (III) share common structural motifs in converting B- to A-DNA.

The (dG)n.(dC)n-containing 34mer DNA duplex [d(A2G15C15T2)]2 can be effectively converted from the B-DNA to the A-DNA conformation by neomycin, spermine and Co(NH3)6(3+). Conversion is demonstrated by a characteristic red shift in the circular dichroism spectra and dramatic NMR spectral changes in chemical shifts. Additional support comes from the substantially stronger CH6/GH8-H3'NOE intensities of the ligand-DNA complexes than those from the native DNA duplex. Such changes are consistent with a deoxyribose pucker transition from the predominate C2'-endo (S-type) to the C3'-endo (N-type). The changes for all three ligand-DNA complexes are identical, suggesting that those three complex cations share common structural motifs for the B- to A-DNA conversion. The A-DNA structure of the 4:1 complex of Co(NH3)6(3+)/d(ACCCGCGGGT) has been analyzed by NOE-restrained refinement. The structural basis of the transition may be related to the closeness of the two negatively charged sugar-phosphate backbones along the major groove in A-DNA, which can be effectively neutralized by the multivalent positively charged amine functions of these ligands. In addition, ligands like spermine or Co(NH3)6(3+) can adhere to guanine bases in the deep major groove of the double helix, as is evident from the significant direct NOE cross-peaks from the protons of Co(NH3)6(3+) to GH8, GH1 (imino) and CH4 (amino) protons. Our results point to future directions in preparing more potent derivatives of Co(NH3)6(3+) for RNA binding or the induction of A-DNA.     

Comparative NMR analysis of the decadeoxynucleotide d-(GCATTAATGC)2 and an analogue containing 2-aminoadenine.

The dodecadeoxynucleotide duplex d-(GCATTAATGC)2 has been prepared with all adenine bases replaced by 2-NH2-adenine. This modified duplex has been characterized by nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific 1H resonance assignments have been obtained by using a variety of 2D NMR methods. Multiple quantum-filtered and multiple quantum experiments have been used to completely assign all sugar ring protons, including 5'H and 5'H resonances. The assignments form the basis for a detailed comparative analysis of the 1H NMR parameters of the modified and parent duplex. The structural features of both decamer duplexes in solution are characteristic of the B-DNA family. The spin-spin coupling constants in the sugar rings and the relative spatial proximities of protons in the bases and sugars (as determined from the comparison of corresponding nuclear Overhauser effects) are virtually identical in the parent and modified duplexes. Thus, substitution by this adenine analogue in oligonucleotides appears not to disturb the global or local conformation of the DNA duplex.     

Conformational variations in d(TGACGTCA) and its reverse sequence d(ACTGCAGT): a joint circular dichroism and nuclear magnetic resonance study

Circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques have been used to characterize the structural properties of the two self-complementary DNA octamers d(TGACGTCA) (I) and d(ACTGCAGT) (II). These display as distinctive features reverse sequences and central steps CpG and GpC, respectively. CD experiments lead to B-form DNA spectra characterized by larger magnitude signals in the case of octamer (I). NMR COSY spectra indicate that in the two octamers all the residues are predominantly south, S, (2'-endo) sugar conformation. NMR NOESY spectra show most of the glycosidic angles confined in the range predicted for B-form DNA although important heterogeneity is noticed along the chains, more pronounced in the case of octamer (I). Both the increase of north, N, (3'-endo) sugar conformation and P (pseudorotation phase angle) deviation from its standard B-form DNA value (162 degrees) express local sequence dependent structure distortions, remarkably visible in CpG step of octamer (I) and agreeing with NOESY cross-peaks intensities. Results interpreted according to Calladine's rules indicate higher cross-chain strains in octamer (I) than in octamer (II). All together, we find evidence to support for octamer (I) in solution local structures with A-DNA properties likely dictated by the central CpG step. Such structures could be involved in the DNA recognition by proteins and anticancerous drugs.     

5-Bromodeoxyuridine radiosensitization: conformation-dependent DNA damage

DNA structure has recently emerged as one of the key factors governing the ability of 5-bromodeoxyuridine (BrdU) to radiosensitize DNA. Here, we report the dependence of the specific damage induced by BrdU for different DNA conformations. Strand breaks are specific for B-form DNA, whereas A-DNA only undergoes formation of piperidine-sensitive DNA lesions. Interstrand cross-links are only found in semi-complementary B-DNA. DNA conformation was altered by gradually rehydrating lyophilized DNA samples, which induces an A- to B-form transition. These results were also validated by irradiating DNA in solution, in the presence or absence of 80% ethanol to induce an A- or B-form, respectively. Alkali-labile DNA lesions were revealed using hot piperidine to transform both base and sugar lesions into strand breaks. We also analyzed the location of damage as a function of DNA structure: piperidine-sensitive lesions were observed exclusively at the site of BrdU substitution, whereas strand breaks were able to migrate along the DNA strand, with a clear preference for the adenine 5' of the BrdU. Thus, not only the hybridization state but also the DNA conformation affect the degree of sensitization by BrdU by influencing the amount and type of damage produced. Although clinical trials using BrdU as a radiosensitizer have been disappointing up to this point, these new findings point to several key features of BrdU radiosensitization that may alter future radiotherapeutic studies.     

Effect of crystal packing environment on conformation of the DNA duplex. Molecular structure of the A-DNA octamer d(G-T-G-T-A-C-A-C) in two crystal forms

The structure of the octamer d(G-T-G-T-A-C-A-C) was determined in two different crystal forms, tetragonal P4(3)2(1)2 and hexagonal P6(1)22. Although in both forms the octamer adopts an A-DNA structure, there are significant conformational differences between them. In particular, the P-05' and the C5'-C4' bonds of the middle adenine (A5) residue exhibit a distorted trans-trans conformation in the tetragonal form, while they adopt the standard gauche-, gauche+ conformation in the hexagonal form. These differences can be correlated with certain features of the crystal packing interactions in the two forms. Furthermore, a comparison of the structures of various A-DNA octamers reveals that the A-form can be divided into two subclasses such that the hexagonal structures have helical and base pair parameters that fall closer to fiber A-DNA values, while in the tetragonal structures these parameters deviate more from fiber A-DNA. These results indicate that environment plays a major role in determining DNA conformation.     

A tandem of SH3-like domains participates in RNA binding in KIN17, a human protein activated in response to genotoxics

The human KIN17 protein is an essential nuclear protein conserved from yeast to human and expressed ubiquitously in mammals. Suppression of Rts2, the yeast equivalent of gene KIN17, renders the cells unviable, and silencing the human KIN17 gene slows cell growth dramatically. Moreover, the human gene KIN17 is up-regulated following exposure to ionizing radiations and UV light, depending on the integrity of the human global genome repair machinery. Its ectopic over-expression blocks S-phase progression by inhibiting DNA synthesis. The C-terminal region of human KIN17 is crucial for this anti-proliferation effect. Its high-resolution structure, presented here, reveals a tandem of SH3-like subdomains. This domain binds to ribonucleotide homopolymers with the same preferences as the whole protein. Analysis of its structure complexed with tungstate shows structural variability within the domain. The interaction with tungstate is mediated by several lysine residues located within a positively charged groove at the interface between the two subdomains. This groove could be the site of interaction with RNA, since mutagenesis of two of these highly conserved lysine residue weakens RNA binding.     

The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).     

Enzymatic removal of ribonucleotides from DNA is essential for mammalian genome integrity and development

The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.     

Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase

The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2′-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.     

Sequence specificity in spermine-induced structural changes in CG-oligomers

The role of spermine in inducing A-DNA conformation in deoxyoligonucleotides has been studied using CCGG and GGCC as model sequences. It has been found that while CCGG adopts an alternating B-DNA conformation in low salt solution at low temperature, addition of spermine to this medium induces a B --greater than A transition. In contrast, the A-DNA-like structure of GGCC in low salt solution at low temperature does not change under the influence of spermine. This suggests a sequence-dependent behaviour of spermine. Further these results suggest that the A-DNA conformation observed in the crystals of d(iCCGG) and d(GGCC)2 might have been due to the presence of spermine in the crystallization cocktail.     

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.