Strand Of Double-stranded DNA Research Articles

Effects of the major formaldehyde catalyzer ADH5 on phenotypes of fanconi anemia zebrafish model.

Fanconi anemia (FA) is a devastating hereditary disorder for which we desperately need a novel therapeutic strategy. It is caused by mutations in one of at least 22 genes in the FA pathway and is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. The FA pathway is required for the efficient repair of damaged DNA, including interstrand cross-links (ICL). Recent studies indicate formaldehyde as an ultimate endogenous cause of DNA damage in FA pathophysiology. Formaldehyde can form DNA adducts as well as ICLs by inducing covalent linkages between opposite strands of double-stranded DNA. In this study, we generated a disease model of FA in zebrafish by disrupting the ube2t or fancd2 gene, which resulted in a striking phenotype of female-to-male sex reversal. Since formaldehyde is detoxified from the body by alcohol dehydrogenase 5 (ADH5), we generated fancd2-/-/adh5-/- zebrafish. We observed a body size reduction and a lower number of mature spermatozoa than wild-type or single knockout zebrafish. To evaluate if increased activity in ADH5 can affect the FA phenotype, we overexpressed human ADH5 in fancd2-/- zebrafish. The progress of spermatogenesis seemed to be partially recovered due to ADH5 overexpression. Our results suggest potential utility of an ADH5 enzyme activator as a therapeutic measure for the clearance of formaldehyde and treatment of FA.

Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links.

DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1–2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above.

Single DNA Translocation and Electrical Characterization Based on Atomic Force Microscopy and Nanoelectrodes.

Precision DNA translocation control is critical for achieving high accuracy in single molecule-based DNA sequencing. In this report, we describe an atomic force microscopy (AFM) based method to linearize a double-stranded DNA strand during the translocation process and characterize the electrical properties of the moving DNA using a platinum (Pt) nanoelectrode gap. In this method, λDNAs were first deposited on a charged mica substrate surface and topographically scanned. A single DNA suitable for translocation was then identified and electrostatically attached to an AFM probe by pressing the probe tip down onto one end of the DNA strand without chemical functionalizations. Next, the DNA strand was lifted off the mica surface by the probe tip. The pulling force required to completely lift off the DNA agreed well with the theoretical DNA adhesion force to a charged mica surface. After liftoff, the captured DNA was translocated at varied speeds across the substrate and ultimately across the Pt nanoelectrode gap for electrical characterizations. Finally, finite element analysis of the effect of the translocating DNA on the conductivity of the nanoelectrode gap was conducted, validating the range of the gap current measured experimentally during the DNA translocation process.

Ultrasensitive hybridization capture: Reliable detection of

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

The Many Roles of Binding Cooperativity in the Control of DNA Replication

The Many Roles of Binding Cooperativity in the Control of DNA Replication

Abstract 4751: APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer

Abstract Introduction: Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide 3A (A3A) is a C to U cytidine deaminase, preferentially deaminating the lagging strand of double-stranded DNA at specific sequence motifs. A3A is overexpressed across multiple tumor types, and in pancreatic ductal adenocarcinoma (PDA), high expression is associated with significantly decreased survival in early stage patients. Large scale sequencing studies of various human tumors (TCGA and ICGC), including PDA, have implicated A3A as a potential driver of tumor mutagenesis. However, the biological relevance of the deamination function of A3A has not yet been shown. Here, we utilize a novel genetically engineered mouse model to show that A3A supports the development of aggressive PDA independent of mutagenic capabilities. Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to dissect the function of A3A on PDA initiation and progression, we made mice with a germline knockin for the coding sequence for human A3A and bred to a well-established GEMM of PDA to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; A3A+/- (KPCY;A3A) mice and compared to KPCY mice. We genotyped tumors from both cohorts using VarScan after exome capture sequencing. Tumor and immune cells were FACS sorted comparative RNAseq performed. The immune contexture of murine and 155 untreated and treated human PDAs was analyzed by performing 7-color multiplex staining and immune cell spatial interaction analysis. Results: All KPCY;A3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p&amp;lt;0.01). One third of the KPCY; A3A mice developed macrometastatic disease compared to 3/12 in the KPCY control. Whole exome sequencing of tumors revealed no significant difference in the number of point mutations or neoantigen load between KPCY;A3A and KPCY tumors. However, histologically, KPCY;A3A tumors contained significantly more desmoplasia and altered immune cell infiltrates. Specifically, comparative RNAseq and IHC analyses revealed dramatic differences in the number and distribution of various T-cell and B-cell subsets in KPCY;A3A mice. Moreover, A3A expression led to abrogation of CD8+ T cell activation and proliferation. Concomitantly, a panel of immune checkpoint-related genes were upregulated in A3A high expressing tumors and cell lines compared to non-A3A expressing controls. This was confirmed in a panel of resected human PDA. Conclusions: Counter to our hypothesis, A3A supports the initiation and growth of PDA in vivo through effects independent of deamination or mutagenesis of cancer cell genomes but rather indirectly through suppression of the tumor immune response. Future studies will address the precise mechanisms underlying A3A-mediated tumor immune evasion. Citation Format: Sonja Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4751.

Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4–5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

Biomolecular Aspects of Second Order Limit Language

The study on the recombinant behavior of double-stranded DNA molecules has led to the mathematical modelling of DNA splicing system. The interdisciplinary study is founded from the knowledge of informational macromolecules and formal language theory. A splicing language is resulted from a splicing system. Recently, second order limit language, a type of the splicing language, has been extensively explored. Before this, several types of splicing languages have been experimentally proven. Therefore, in this paper, a laboratory experiment was conducted to validate the existence of a second order limit language. To accomplish it, an initial strand of double-stranded DNA, amplified from bacteriophage lambda, was generated through polymerase chain reaction to generate thousands of copies of double-stranded DNA molecules. A restriction enzyme and ligase were added to the solution to complete the reaction. The reaction mixture was then subjected to polyacrylamide gel electrophoresis to separate biological macromolecules according to their sizes. A mathematical model derived at the early study was used to predict the approximate length of each string in the splicing language. The results obtained from the experiment are then used to verify the mathematical model of a second order limit language. This study shows that the theory on the second order limit language is biologically proven hence the model has been validated.

T7 helicase unwinding and stand switching investigated via single-molecular technology

Single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers are widely used to study the molecular motors because of their high resolution and real-time observation. In this work, we choose these two techniques as the research means. The bacteriophage T7 helicase, as the research object, serves as a model protein for ring-shaped hexameric helicase that couples deoxythymidine triphosphate (dTTP) hydrolysis to unidirectional translocation. The DNA strand separation is 5'-3'-along one strand of double-stranded DNA. Using smFRET and magnetic tweezers to study the unwinding process of T7 helicase, we can have more in depth understanding of the unwinding and strand switching mechanisms of the ring-shaped hexameric helicases. First, by designing DNA substrates with different 3'-tail structures, we find that the 3'-tail is required for T7 helicase unwinding process, no matter whether it is single-stranded or double-stranded. These results confirm an interaction between T7 helicase and 3'-tail. Second, examining the dependence of unwinding process on GC content in DNA sequence, we find that as GC content increases, T7 helicase has higher chances to stop and slips back to the initial position by annealing stress or dissociating from DNA substrate. As the GC content increases to 100%, 79% helicases could not finish the unwinding process. Third, by further analysing the experimental data, two different slipping-back phenomena of T7 helicase are observed. One is instantaneous and the other is slow. The results from the experiment on magnetic tweezers also confirm this slow slipping-back phenomenon. This instantaneous slipping-back results from the rewinding process of unwound single-stranded DNA as studied previously. When T7 helicase cannot continue unwinding because of the high GC content in DNA sequence, it dissociates from the single-stranded DNA or slips back to the initial position very quickly because of the annealing stress. However, this slow slipping-back phenomenon cannot be explained by this reason. According to previous researches, T7 helicase can only be translocated or unwound from 5' to 3' along one strand of double-stranded DNA because of the polarity principle. We suggest that this slow slipping-back is induced by the strand switching process of T7 helicase. Through this strand switching process, T7 helicase binds to the 3'-strand and are translocated along it from 5' to 3' to the initial position, results in this slow slipping-back phenomenon. This is the first time that the slow slipping-back phenomenon has been observed, which strongly suggests the strand switching process of T7 helicase. Based on our results and previous researches, we propose the model of this strand switching process and this model may be extended to all ring-shaped hexameric helicases.

Use of the Cy3 and Cy5 Fluorescent Labels to Protect a DNA Strand from Degradation under λ Exonuclease Treatment

λ Exonuclease hydrolyzes a 5′-phosphorylated strand of double-stranded DNA in the 5′–3′ direction. In this paper, the activity of the enzyme with respect to DNA substrates containing Cy3 and Cy5 fluorescent labels at their 5′ ends is studied for the first time. It is demonstrated using the fluorescence procedure for measuring the exonuclease activity that double-stranded DNA, of which both the 5′ ends contain these fluorophores, is not destroyed under the action of λ exonuclease. Using the electrophoretic separation of DNA in polyacrylamide gel, the process of obtaining single-stranded DNA from double-stranded precursors containing different labels at the 5′ ends is studied. It is shown that the introduction of Cy3 and Cy5 fluorophores to the 5′ end of the DNA strand protects this strand from enzyme damage both in the duplex and in single-stranded form, and these labels can be used to produce fluorescently labeled single-stranded DNA.

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5′-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria.

Backbone Dynamics Study of the Zβ Domain of Human DAI Bound to Z‐DNA#

Innate immune response is an essential defense against infections. Human DNA-dependent activator of IFN-regulatory factor (DAI; also known as ZBP1 or DLM-1) protein activates the innate immune response in response to DNA. The DAI protein contains two tandem Z-DNA binding domains (Zα and Zβ) at the N-terminus. The two Z-DNA binding domains are thought to be essential for full activation of a DNA-dependent immune response in vivo. The crystal structure of the Zα domain of human ADAR1 (ZαADAR1) in complex with Z-DNA shows that two Zα domains bind to each strand of double-stranded DNA and have twofold symmetry with respect to the DNA's helical axis. Structural studies with the Zβ domain of human DAI (ZβDAI) in complex with Z-DNA suggest that the ZβDAI structure is similar to that of ZαADAR1, although it demonstrates an unusual Z-DNA recognition. A previous NMR study of the ZβDAI complexed with a six-base-pair (6-bp) DNA duplex, d(CG)3, suggests that ZβDAI binds to Z-DNA via an activedi B–Z transition mechanism, where two ZβDAI proteins bind to B-DNA to form the ZβDAI–B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. To investigate the structural and dynamic properties of ZβDAI when it binds toDNAduplex and induces the B–Z transition in a DNA duplex, we have performed NMR backbone dynamics experiments on the free ZβDAI and the ZβDAI–d (CG)3 complex. The results revealed that ZβDAI exhibits a distinct change in the dynamics during theB–Z transition ofDNA duplex compared to ZαADAR1. This study provides valuable insights into the molecular mechanism of the B–Z transition induced by ZβDAI.

Electronic states of DNA and M-DNA studied by optical absorption.

To unveil the electronic states of divalent metal ion incorporated M-DNAs, where M is Mg, Mn, Ni, Co, or Fe, optical absorption spectra have been studied in aqueous solutions of single-stranded (SS) 30 mer DNA of poly(dA) (adenine), poly(dG) (guanine), poly(dT) (thymine), poly(dC) (cytosine), salmon-sperm DNA (B-DNA), and M-DNA. The absorption spectrum of the double-stranded (DS) B-DNA can be reproduced with the sum of the four absorption spectra of the SS oligo-DNAs in the ratio corresponding to the composition of B-DNA. This observation suggests that the interactions between complementary strands of DS DNA are negligibly weaker than the bandwidths of the optical spectra. In the metal-incorporated M-DNAs, except for Fe-DNA, the absorption spectra show no significant qualitative change from that of B-DNA. Quantitatively, however, the absorption intensity decreases by ≈ 15% uniquely in a DS poly(dA)-poly(dT) solution with adding MCl(2), while nothing happens quantitatively and qualitatively in any SS oligo-DNA and DS poly(dG)-poly(dC) solutions, suggesting some suppression of the electronic excitation only in the Adenine-M-Thymine complex. In contrast, remarkable differences have been observed in Fe-DNA, prepared with FeCl(2) and B-DNA. New absorption bands appear in the intragap energy of Fe-DNA, in addition to the suppression of the interband absorption peak of DNA at 4.8 eV. The intragap absorption is attributed to the appearance of Fe(3+) species with the same spectral feature as that of FeCl(3), that is, purely ionic Fe(3+) species. This observation suggests that FeCl(2) + B-DNA forms Fe-DNA with hydrated Fe(3+) ions with ionic bonds. Thus, it is concluded that the charge transfer from Fe(2+) to DNA has occurred in Fe-DNA and that the transferred charges are expected to be located in the nearby bases.

Massively parallel characterization of restriction endonucleases

Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site–flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.

Patterns of nucleotide asymmetries in plant and animal genomes

Symmetry in biology provides many intriguing puzzles to the scientist's mind. Chargaff's second parity rule states a symmetric distribution of oligonucleotides within a single strand of double-stranded DNA. While this rule has been verified in a wide range of microbial genomes, it still awaits explanation. In our study, we inquired into patterns of mono- and trinucleotide intra-strand parity in complex plant genomic sequences that became available during the last few years, and compared these to equally complex animal genomes. The degree and patterns of deviation from Chargaff's second rule were different between plant and animal species. We observed a universal inter-chromosomal homogeneity of mononucleotide skews in coding sequences of plant chromosomes, while the base composition of animal coding sequences differed between chromosomes even within a single species. We also found differences in the base composition of dicot introns in comparison to those of monocots. These genome-wide patterns were limited to genic regions and were not encountered in inter-genic sequences. We discuss the implications of our findings in relation to hypotheses about functional correlations of intra-strand parity which have hitherto been put forward. Furthermore, we propose more recent polyploidization and subsequent homogenization of homoeologues as a possible reason for more homogeneous skew patterns in plants.

Nanophotonic Control of the Förster Resonance Energy Transfer Efficiency

We have studied the influence of the local density of optical states (LDOS) on the rate and efficiency of Förster resonance energy transfer (FRET) from a donor to an acceptor. The donors and acceptors are dye molecules that are separated by a short strand of double-stranded DNA. The LDOS is controlled by carefully positioning the FRET pairs near a mirror. We find that the energy transfer efficiency changes with LDOS, and that, in agreement with theory, the energy transfer rate is independent of the LDOS, which allows one to quantitatively control FRET systems in a new way. Our results imply a change in the characteristic Förster distance, in contrast to common lore that this distance is fixed for a given FRET pair.

Escherichia coli topoisomerase I is an iron and zinc binding protein

Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV-Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.

Population Analysis of the Intermediate Complex States During B-Z Transition of Non-CG-repeat DNA Duplexes Induced by the Zα Domain of Human ADAR1

Figure 2. (A) The kex values of the G4b imino proton for the CA6ZαADAR1 complex determined at 25 C and (B) kex values of the G2b imino proton for the TA6-ZαADAR1 complex determined at 15 C as a function of the fZ. Black solid lines are the best fit to Eq. 1, where the kex data were weighted by the inverse of their variance. The grey lines indicate their upper and lower confidence limits (95% confidence level). Z-DNA contains nucleic acid bases in alternating antiand syn-conformations along the nucleotide chain and has only one groove that is similar to the minor groove of B-DNA. Z-DNA is in a higher energy conformation than B-DNA and is stabilized by negative supercoiling generated in vivo. Human ADAR1 has two left-handed Z-DNA binding domains at its NH2-terminus, Zα and Zβ, preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The co-crystal structure of the Zα domain of human ADAR1 (ZαADAR1) bound to Z-DNA revealed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with two-fold symmetry with respect to the DNA helical axis. A structural study showed that ZαADAR1 binds to the Z-conformation of non-CG-repeat DNA duplexes through a common structural feature rather than by a specific sequence or structural alternations. A previous NMR study on a d(CGCGCG)2-ZαADAR1 complex suggests an active-mono B-Z transition mechanism (see Fig. 1) in which the ZαADAR1 protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second ZαADAR1 molecule. Recently, we have reported NMR hydrogen exchange data of complexes between ZαADAR1 and the non-CG-repeat DNA duplexes, d(CACGTG)2 [referred to as CA6] or d(CGTACG)2 [referred to as TA6], with a variety of protein-to-DNA (P/N) molar ratios. The kex data for the G4b of the CA6-ZαADAR1 complex and for the G2b of the TA6-ZαADAR1 complex showed significant changes as the Z-DNA fraction (fZ) was increased (meaning that the P/N ratio increased) (see Fig. 2). These changes of the kex data can be explained by the presence of mixtures of two imino protons from B-form DNA (referred to as B) and B-DNA-ZαADAR1 complex (referred to as BP) in the imino peaks as given by Eq. 1:

NMR Spectroscopic Elucidation of the B−Z Transition of a DNA Double Helix Induced by the Zα Domain of Human ADAR1

The human RNA editing enzyme ADAR1 (double-stranded RNA deaminase I) deaminates adenine in pre-mRNA to yield inosine, which codes as guanine. ADAR1 has two left-handed Z-DNA binding domains, Z alpha and Z beta, at its NH(2)-terminus and preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The cocrystal structure of Z alpha(ADAR1) complexed to Z-DNA showed that one monomeric Z alpha(ADAR1) domain binds to one strand of double-stranded DNA and a second Z alpha(ADAR1) monomer binds to the opposite strand with 2-fold symmetry with respect to DNA helical axis. It remains unclear how Z alpha(ADAR1) protein specifically recognizes Z-DNA sequence in a sea of B-DNA to produce the stable Z alpha(ADAR1)-Z-DNA complex during the B-Z transition induced by Z alpha(ADAR1). In order to characterize the molecular recognition of Z-DNA by Z alpha(ADAR1), we performed circular dichroism (CD) and NMR experiments with complexes of Zalpha(ADAR1) bound to d(CGCGCG)(2) (referred to as CG6) produced at a variety of protein-to-DNA molar ratios. From this study, we identified the intermediate states of the CG6-Z alpha(ADAR1) complex and calculated their relative populations as a function of the Z alpha(ADAR1) concentration. These findings support an active B-Z transition mechanism in which the Z alpha(ADAR1) protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second Z alpha(ADAR1) molecule.

Force Calculations for DNA-PAMAM Dendrimer Interactions from Molecular Dynamics Simulations

Polyamidoamine (PAMAM) dendrimers are known to bind and condense plasmid DNA (1,2,3). However, the nature of the interaction between dendrimers and DNA and the mechanism of compaction is not fully understood. Potentials of mean force and forces of interaction were calculated from all-atom umbrella sampling simulations of amine-terminated G3 dendrimers and a 24bp strand of double-stranded DNA. Our simulations show that dendrimers and DNA interact with each other over large distances. Simulations also reveal that even low-generation dendrimers can induce significant bending in DNA and that the dendrimer also deforms considerably upon interaction with the DNA. We compare forces calculated from these simulations with optical tweezer experiments on DNA condensation by dendrimers (3) and propose an explanation for the compaction of DNA by dendrimers observed at forces over 60pN.1. A.U. Bielinska, J.F. Kukowska-Latallo, J.R. Baker, Gene Structure and Expression, 1353, 180 (1997).2. W. Chen, N.J. Turro, D.A. Tomalia, Langmuir, 16, 15 (2000).3. F. Ritort, S. Mihardja. S.B. Smith, C. Bustamante, Phys. Rev. Letters, 96, 118301 (2006).