DNA Cleavage Sites Research Articles

Key role of the REC lobe during CRISPR-Cas9 activation by 'sensing', 'regulating', and 'locking' the catalytic HNH domain.

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 – a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution – reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1–3 regions ‘sense’ nucleic acids, ‘regulate’ the HNH conformational transition, and ultimately ‘lock’ the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1–3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

The Sequence Preference of Gamma-Radiation-Induced Damage in End-Labeled DNA after Heat Treatment.

In this work, we examined the DNA sequence preference of gamma-radiation-induced DNA damage in purified DNA sequences after heat treatment. DNA was fluorescently end-labeled and gamma-radiation-induced DNA cleavage was examined using capillary electrophoresis with laser-induced fluorescence detection. Our findings provide evidence that gamma-radiation-induced DNA damage to end-labeled DNA is nonrandom and has a sequence preference. The degree of cleavage was quantified at each nucleotide, and we observed that preferential cleavage occurred at C nucleotides with lesser cleavage at G nucleotides, while being very low at T nucleotides. The differences in percentage cleavage at individual nucleotides ranged up to sixfold. The DNA sequences surrounding the most intense radiation-induced DNA cleavage sites were examined and a consensus sequence 5'-AGGC*C (where C* is the cleavage site) was found. The highest intensity gamma-radiation-induced DNA cleavage sites were found at the dinucleotides, 5'-GG*, 5'-GC*, 5'-C*C and 5'-G*G and at the trinucleotides, 5'-GG*C, 5'-TC*A, 5'-GG*G and 5'-GC*C. These findings have implications for our understanding of ionizing radiation-induced DNA damage.

Structural insights into DNA cleavage activation of CRISPR-Cas9 system

CRISPR-Cas9 technology has been widely used for genome engineering. Its RNA-guided endonuclease Cas9 binds specifically to target DNA and then cleaves the two DNA strands with HNH and RuvC nuclease domains. However, structural information regarding the DNA cleavage-activating state of two nuclease domains remains sparse. Here, we report a 5.2 Å cryo-EM structure of Cas9 in complex with sgRNA and target DNA. This structure reveals a conformational state of Cas9 in which the HNH domain is closest to the DNA cleavage site. Compared with two known HNH states, our structure shows that the HNH active site moves toward the cleavage site by about 25 and 13 Å, respectively. In combination with EM-based molecular dynamics simulations, we show that residues of the nuclease domains in our structure could form cleavage-compatible conformations with the target DNA. Together, these results strongly suggest that our cryo-EM structure resembles a DNA cleavage-activating architecture of Cas9.

Design, synthesis and biological evaluation of 1,3-diphenylbenzo[f][1,7]naphthyrdines

A novel series of twenty 1,3-diphenylbenzo[f][1,7]benzonaphthyrdine derivatives were designed and synthesized through intermolecular imino Diels-Alder reaction. Their in vitro cytotoxic activities were evaluated against six human cancer cell lines (NCIH23, HCT15, NUGC-3, ACHN, PC-3, and MDA-MB-231). Majority of synthesized compounds exhibited significant cytotoxic activities against all tested human cancer cell lines. Among them 4l, 4m, and 4o derivatives exhibited most promising cytotoxic activities. Furthermore these compounds were evaluated against human Topoisomerase IIα inhibition. Interestingly, the compound 4l exhibited 1.3 and 1.2 times more potent human Topoisomerase IIα inhibition than the reference drug etoposide in both 100µM and 20µM concentrations respectively. Molecular docking studies for the compound 4l have also been executed by Sybyl X-2.1 in which it reveals the binding site of the compound 4l with topo IIα DNA cleavage site where etoposide was situated. The benzo[f][1,7]naphthyridine ring was stacked between the DNA bases of the cleavage site.

Mitochondria-targeted nanocarriers using hyperbranched polycations wrapped carbon nanotubes for augment photodynamic therapeutic effects

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.

Effect of NaeI-L43K mutation on protein dynamics and DNA conformation: Insights from molecular dynamics simulations

Protein-DNA interactions are an important class of biomolecular interactions inside the cell. Delineating the mechanisms of protein-DNA interactions and more specifically, how proteins search and bind to their specific cognate sequences has been the quest of many in the scientific community. Restriction enzymes have served as useful model systems to this end. In this work, we have investigated using molecular dynamics simulations the effect of L43K mutation on NaeI, a type IIE restriction enzyme. NaeI has two domains, the Topo and the Endo domains, each binding to identical strands of DNA sequences (GCCGGC)2. The binding of the DNA to the Topo domain is thought to enhance the binding and cleavage of DNA at the Endo domain. Interestingly, it has been found that the mutation of an amino acid that is distantly-located from the DNA cleavage site (L43K) converts the restriction endonuclease to a topoisomerase. Our investigations reveal that the L43K mutation not only induces local structural changes (as evidenced by changes in hydrogen bond propensities and differences in the percentage of secondary structure assignments of the residues in the ligase-like domain) but also alters the overall protein dynamics and DNA conformation which probably leads to the loss of specific cleavage of the recognition site. In a larger context, our study underscores the importance of considering the role of distantly-located amino acids in understanding protein-DNA interactions.

Genome Organization Drives Chromosome Fragility

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.

Endonuclease domain of non-LTR retrotransposons: loss-of-function mutants and modeling of the R2Bm endonuclease.

Non-LTR retrotransposons are an important class of mobile elements that insert into host DNA by target-primed reverse transcription (TPRT). Non-LTR retrotransposons must bind to their mRNA, recognize and cleave their target DNA, and perform TPRT at the site of DNA cleavage. As DNA binding and cleavage are such central parts of the integration reaction, a better understanding of the endonuclease encoded by non-LTR retrotransposons is needed. This paper explores the R2 endonuclease domain from Bombyx mori using in vitro studies and in silico modeling. Mutations in conserved sequences located across the putative PD-(D/E)XK endonuclease domain reduced DNA cleavage, DNA binding and TPRT. A mutation at the beginning of the first α-helix of the modeled endonuclease obliterated DNA cleavage and greatly reduced DNA binding. It also reduced TPRT when tested on pre-cleaved DNA substrates. The catalytic K was located to a non-canonical position within the second α-helix. A mutation located after the fourth β-strand reduced DNA binding and cleavage. The motifs that showed impaired activity form an extensive basic region. The R2 biochemical and structural data are compared and contrasted with that of two other well characterized PD-(D/E)XK endonucleases, restriction endonucleases and archaeal Holliday junction resolvases.

Preferential Localization of Iron in The Chromatin of Fe-Enriched Cells Is Linked to DNA Cleavage Sites and Control of Carcinogenesis

DNA oxidation by oxygen-radicals generated via an iron catalyzed Fenton reaction has been extensively investigated, but little is known about iron localization in the nuclei of mammalian cells. In vitro studies showed the presence of five oxygen and one nitrogen atoms in the inner coordination sphere of the Fe(II)-DNA complex using X-ray absorption spectroscopy (XANES and EXAFS). The identification of ferritin in the nucleus of cultured cells, as well as iron-protein receptors in the nuclear membrane suggests that iron is actively transported into the nucleus. Therefore, pictures from energy-loss spectroscopic imaging (ESI) are included in this mini-review to illustrate the distribution of iron in a fibroblast cell line. These fibroblasts were iron-overloaded by being cultured in a medium containing Fe(III)-nitrilotriacetate (FeNTA). The elemental mapping of iron and phosphorus was coincident in ultrastructures and revealed a significant concentration of both in condensed chromatin; by contrast, the elemental mapping of nitrogen, used as a control, revealed a homogenous distribution across the entire cell. This observed preferential localization was surprising, considering the pro-oxidant status of iron and the importance of maintaining genome integrity. Interestingly, recent published works demonstrates that iron chelators such as genistein and daidzein, derived from soy isoflavones, can attenuate the expression of genes related to increased cancer risk and oxidative damage in the reproductive tract of female mice. In this same sense, mesenchymal stem cells also exhibit antioxidant properties by reducing superoxide, lipid peroxidation, and DNA breaks to healthful levels after transplantation into the brains of stroke prone spontaneously hypertensive rats. Therefore, we bring attention to the use of all potential antioxidants, particularly those with affinity for both iron and DNA, in therapies for several diseases.

Abstract 4517: Plasma and tumor pharmacokinetics of IV LMP744, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study

Abstract Introduction: LMP744 (NSC706744) is one of 3 indenoisoquinolines being evaluated in pet dogs with lymphoma to define their safety, pharmacokinetics, and pharmacodynamic modulation (COTC007b). LMP744 forms stable DNA-topoisomerase I (Top1) cleavage complexes and induces unique DNA cleavage sites relative to approved Top1 poisons; LMP744 is not a substrate for ABC transporters and does not have the stability issues inherent to camptothecins. Here we report the plasma and tumor pharmacokinetics of LMP744 administered IV daily x 5 to patient dogs. Methods: Eligibility included: dogs >15 kg with histologically confirmed non-Hodgkin's lymphoma with nodal presentation (stage 2 or greater) and minimal nodes size for biopsy of 3 cm in the longest dimension, performance status of Grade 0 or 1 and informed owner consent. Dose levels (DL) explored were 25, 50, 75, 100, and 125 mg/m2/day through a 3+3 dose escalation study design. LMP744 was administered over 1 h IV daily x 5, Q28D. LMP744 was quantitated with a validated LC-MS/MS assay, and plasma pharmacokinetic parameters determined non-compartmentally with PK Solutions and plasma and tumor parameters compartmentally with ADAPT5 through iterated two stage (ITS) and maximum likelihood solution with the expectation maximization algorithm (MLEM). Results: All 18 dogs had useable pharmacokinetic data. Non-compartmental analysis suggested linear relationships between plasma Cmax and tumor C2h vs. dose. LMP744 distributed extensively to tumor tissue and mean tumor concentrations were at least 2 orders of magnitude higher than plasma concentrations at equivalent time points of 2, 6 and 120 h. Compartmental modeling with a 3 compartment linear model resulted in a good fit to the plasma data. The parameters and%CVs respectively for Clt, Vc, Cld1, Vp1, Cld2, Vp2 were 57.5 L/h/m2 (29%), 136 L/m2 (24%), 234 L/h/m2 (24%), 1180 L/m2 (27%), 30.8 L/h/m2 (63%), 5140 L/m2 (67%). Sequential plasma half-lives were 16.5 min, 13 h, and 183 (31%) h, respectively. In line with the long t1/2, plasma accumulation was observed on this daily times-5 schedule. Conclusion: The plasma pharmacokinetics of LMP744 in dogs display triphasic behavior, a relatively high total body clearance at 57.5 L/h/m2, and a terminal half-life of 183 h. The favorable biodistribution to tumor may be a valuable distinction to consider in the translation of this novel indenoisoquinoline to the clinic. Support: N01-CM-2011-00015, N01-CM-42202 and P30-CA-47904 Note: This abstract was not presented at the meeting. Citation Format: Julie L. Eiseman, Julianne Holleran, David L. McCormick, Miguel Muzzio, Joseph M. Covey, Chand Khanna, Christina Mazcko, Yves Pommier, Melissa Paolini, Amy Leblanc, Jenna H. Burton, James H. Doroshow, Joseph E. Tomaszewski, Jan H. Beumer. Plasma and tumor pharmacokinetics of IV LMP744, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2015-4517

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

BackgroundHigh-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data.MethodsIn this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome.ResultsWe show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA.ConclusionsThese background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0107-z) contains supplementary material, which is available to authorized users.

Novel copper(II) complexes of p-tert-butylcalix[4]arene diamide derivatives: synthesis, antimicrobial and DNA cleavage activities

In this study, two new Cu(II) complexes of p-tert-butylcalix[4]arene amide derivatives (8, 9) have been synthesised and investigated their DNA cleavage and antimicrobial activities. On the basis of spectral studies, a smaller distortion of square planar geometry has been proposed for both of the copper(II) complexes. The DNA cleavage studies show that the ligands are not efficacious, whereas the complexes have high activity. Futhermore, in order to determine the site of DNA cleavage, the DNA interactions of these compounds were investigated with some restriction enzymes. In addition, all synthesised compounds were screened for antimicrobial activities against some bacteria and for antifungal activities against yeast strains. The results showed that ethyl ester and furfuryl amide derivatives of the calixarenes are more efficient than other compounds against tested bacteria. However, complexes have not been effective. In case of DNA interaction studies, compounds were very effective against plasmid DNA.

Abstract 4632: Plasma and tumor pharmacokinetics of IV LMP400, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study

Abstract Introduction: LMP400 is one of 3 indenoisoquinolines that are being evaluated in pet dogs with lymphoma to define their safety, pharmacokinetics, and pharmacodynamic modulation (COTC007b). LMP400 forms stable DNA-topoisomerase I (Top1) cleavable complexes and induces unique DNA cleavage sites relative to approved Top1 poisons; LMP400 is not a substrate for ABC transporters and does not have the stability issues that most camptothecin analogs do. Here we report the plasma and uncoupled tumor pharmacokinetics of LMP400 administered IV daily x 5 to patient dogs. Methods: Eligibility included: dogs >15 kg with histologically confirmed non-Hodgkin's lymphoma with nodal presentation (stage 2 or greater) and minimal nodes size for biopsy of 3 cm in the longest dimension, performance status of Grade 0 or 1 and informed owner consent. Dose levels (DL) explored were 8, 16, 24, and 40 mg/m2/day through a 3+3 dose escalation study design. LMP400 was administered over 1 h IV daily x 5, Q28D. LMP400 was quantitated with a validated LC-MS/MS assay (PMID: 20236781), and plasma pharmacokinetic parameters determined non-compartmentally with PK Solutions and plasma and tumor parameters compartmentally with ADAPT5 through iterated two stage (ITS) and maximum likelihood solution with the expectation maximization algorithm (MLEM). Results: All fifteen dogs had useable pharmacokinetic data. Non-compartmental analysis suggested linear relationships between plasma Cmax and AUC vs. dose and tumor C2h vs. dose. Plasma terminal half-life was 11.9 ± 5.0 h and the CL was 26.7 L/h/m2. In line with the t1/2, slight plasma accumulation was observed on this daily times 5 schedule. Compartmental modeling with a 2 compartment linear model resulted in a good fit to the data. The parameters and %CVs respectively for Ke, Vc, Kcp, Kpc, Kct and Ktc were 0.181 h-1 (42.6%), 108 L/m2 (32.7%), 0.250 h-1 (30.1%), 0.0602 h-1 (67%), 0.0632 h-1 (3.65%), and 0.649 h-1 (42.2%). Plasma compartmental CL and half-life were 19.5 L/h/m2 and 29.7 h. Conclusion: The plasma pharmacokinetics of LMP400 in dogs display biphasic behavior, a relatively high total body clearance at 19.5 L/h/m2, and a terminal half-life of 29.7 h. LMP400 distributed to the tumors and mean tumor concentrations were at least 10-fold higher than plasma concentrations at equivalent time points of 2, 6 and 120 h. This favorable biodistribution to tumor (lymph node) may be a valuable distinction to consider in the translation of this novel indenoisoquinoline to the clinic. Support: N01-CM-2011-00015, N01-CM-42202 and P30-CA-47904 Citation Format: Julie L. Eiseman, Julianne Holleran, David L. McCormick, Miguel Muzzio, Joseph M. Covey, Chand Khanna, Christina Mazcko, Yves Pommier, Melissa Paoloni, James D. Doroshow, Joseph E. Tomaszewski, Jan H. Beumer. Plasma and tumor pharmacokinetics of IV LMP400, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2014-4632

Abstract 4643: Phase I pharmacokinetics and pharmacodynamics of a novel indenoisoquinoline topoisomerase 1 (TOP1) inhibitor, LMP400, administered on a daily x 5 schedule

Abstract Introduction: Indenoisoquinoline LMP 400: 1) forms stable DNA-TOP1 cleavage complexes; 2) binds to unique DNA cleavage sites relative to approved top1 poisons; 3) is not a substrate for ABC transporters. Here we report the plasma pharmacokinetics, pharmacodynamics and optimal sampling of LMP400 administered IV daily x 5 in a phase I trial (NCI#8273). Methods: Eligibility included: refractory solid tumors or lymphoma, ≥18 yrs old; KPS > 70%; adequate organ function. Dose levels (DL) explored were 2.5, 5, 10, 20, 40, 60 and 80 mg/m2/day, following an accelerated titration design transitioning to 3+3 design after one DLT or 2 gr 2 toxicities during cycle 1. LMP400 was administered over 1 h iv daily x 5, q28d. LMP400 was quantitated with a validated LC-MS/MS assay (PMID: 20236781), and pharmacokinetic parameters determined non-compartmentally with PK Solutions and compartmentally with ADAPT5 through iterative two-stage (ITS) and maximum likelihood expectation maximization (MLEM). Exposure metrics were correlated with decreases in neutrophil counts. Optimal sampling strategies were explored with D-optimality. Results: Twenty subjects had useable pharmacokinetic data. Non-compartmental analysis suggested linear relationships between Cmax and AUCobs vs dose. Given the long half-life and daily administration, substantial plasma accumulation was observed. Compartmental modeling with a 2-compartment linear model resulted in a good fit to the data. Population means (population CV%) of CLt, Vc, CLd, and Vp were 1.54 (24.9%) L/h/m2, 29.7 (35.3%) L/m2, 11.4 (35.8%) L/h/m2, and 66.3 (34.7%) L/m2. Distribution and terminal elimination half-lives were 1.17 (25.9%) h and 46.0 (36.3%) h. %decrease in neutrophil counts was correlated with AUC and C24h. C24h was highly correlated with AUC (R2=0.92). Optimal sampling to determine CLt (expected SE 7.96%) required a single sample at 168 h (day 8). To determine all parameters with an expected SE<25%, and limiting samples to within 6 h on the first day, the following 5 time points were optimal: 1) 15 min into day 1 infusion, 2+3) approximately 2.3 h after day 1 infusion [2 samples], 4) day 2 trough before infusion, and 5) 168 h. Conclusion: LMP400 displays biphasic plasma pharmacokinetic behavior, a relatively low total body clearance at 1.54 L/h/m2, and a long terminal half-life at 46 h. Exposure was correlated with %decrease in neutrophils, the DLT of LMP400. The optimal sampling times will aid the design of future phase II studies aimed at further documenting the exposure response relationships of LMP400. The long half-life and accumulation of LMP400 in plasma led to the initiation of a phase I study where LMP400 is dosed once weekly. These studies should further define which exposure metric best correlates with toxicity and anti-tumor activity. Support: N01-CM-2011-00015, U01-CA-099168, P30-CA-47904 Citation Format: Jan H. Beumer, Julianne Holleran, James Doroshow, Alice Chen, Deborah Allen, Joseph Covey, Joseph Tomaszewski, Yves Pommier, Shivaani Kumar, Julie L. Eiseman. Phase I pharmacokinetics and pharmacodynamics of a novel indenoisoquinoline topoisomerase 1 (TOP1) inhibitor, LMP400, administered on a daily x 5 schedule. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4643. doi:10.1158/1538-7445.AM2014-4643

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

A short DNA sequence confers strong bleomycin binding to hairpin DNAs.

Bleomycins A5 and B2 were used to study the structural features in hairpin DNAs conducive to strong BLM–DNA interaction. Two members of a 10-hairpin DNA library previously found to bind most tightly to these BLMs were subsequently noted to share the sequence 5′-ACGC (complementary strand sequence 5′-GCGT). Each underwent double-strand cleavage at five sites within, or near, an eight base pair region of the DNA duplex which had been randomized to create the original library. A new hairpin DNA library was selected based on affinity for immobilized Fe(III)·BLM A5. Two of the 30 newly identified DNAs also contained the sequence 5′-ACGC/5′-GCGT. These DNAs bound to the Fe(II)·BLMs more tightly than any DNA characterized previously. Surface plasmon resonance confirmed tight Fe(III)·BLM B2 binding and gave an excellent fit for a 1:1 binding model, implying the absence of significant secondary binding sites. Fe(II)·BLM A5 was used to assess sites of double-strand DNA cleavage. Both hairpin DNAs underwent double-strand cleavage at five sites within or near the original randomized eight base region. For DNA 12, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA 13 underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA 14, which bound Fe·BLM poorly, was converted to a strong binder (DNA 15) by insertion of the sequence 5′-ACGC/5′-GCGT. These findings reinforce the idea that tighter DNA binding by Fe·BLM leads to increased double-strand cleavage by a novel mechanism and identify a specific DNA motif conducive to strong BLM binding and cleavage.

Molecular docking studies of curcumin natural derivatives with DNA topoisomerase I and II-DNA complexes.

DNA topoisomerase I (topo I) and II (topo II) are essential enzymes that solve the topological problems of DNA by allowing DNA strands or double helices to pass through each other during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Their critical roles make topoisomerases an attractive drug target against cancer. The present molecular docking study provides insights into the inhibition of topo I and II by curcumin natural derivatives. The binding modes suggested that curcumin natural derivatives docked at the site of DNA cleavage parallel to the axis of DNA base pairing. Cyclocurcumin and curcumin sulphate were predicted to be the most potent inhibitors amongst all the curcumin natural derivatives docked. The binding modes of cyclocurcumin and curcumin sulphate were similar to known inhibitors of topo I and II. Residues like Arg364, Asn722 and base A113 (when docked to topo I-DNA complex) and residues Asp479, Gln778 and base T9 (when docked to topo II-DNA complex) seem to play important role in the binding of curcumin natural derivatives at the site of DNA cleavage.

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes: SOLUTION STRUCTURE AND METAL ION BINDING SITES OF THE RNA·DNA COMPLEX

Group II intron ribozymes catalyze the cleavage of (and their reinsertion into) DNA and RNA targets using a Mg2(+)-dependent reaction. The target is cleaved 3' to the last nucleotide of intron binding site 1 (IBS1), one of three regions that form base pairs with the intron's exon binding sites (EBS1 to -3).We solved the NMR solution structure of the d3' hairpin of the Sc.ai5γ intron containing EBS1 in its 11-nucleotide loop in complex with the dIBS1 DNA 7-mer and compare it with the analogous RNA-RNA contact. The EBS1-dIBS1 helix is slightly flexible and non-symmetric. NMR data reveal two major groove binding sites for divalent metal ions at the EBS1-dIBS1 helix, and surface plasmon resonance experiments show that low concentrations of Mg2(+) considerably enhance the affinity of dIBS1 for EBS1. Our results indicate that identification of both RNA and DNA IBS1 targets, presentation of the scissile bond, and stabilization of the structure by metal ions are governed by the overall structure of EBS1-dIBS1 and the surrounding loop nucleotides but are irrespective of different EBS1-(d)IBS1 geometries and interstrand affinities.

The I-TevI Nuclease and Linker Domains Contribute to the Specificity of Monomeric TALENs

Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5′-CNNNG-3′. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints.

Design, synthesis and systematic evaluation of cytotoxic 3-heteroarylisoquinolinamines as topoisomerases inhibitors

A series of 3-heteroarylisoquinolinamines were designed, synthesized and evaluated for cytotoxicity, topoisomerases (topos) inhibitory activities and cell cycle inhibition. Several of the 3-heteroarylisoquinolines exhibited selective cytotoxicity against human ductal breast epithelial tumor (T47D) cells over non-cancerous human breast epithelial (MCF-10A) and human prostate cancer (DU145) cells. Most of the derivatives showed greater cytotoxicity in human colorectal adenocarcinoma (HCT-15) cells than camptothecin (CPT), etoposide and doxorubicin (DOX). Generally, 3-heteroarylisoquinolinamines displayed greater affinity for topo I than topo II. 3-Heteroarylisoquinolinamines with greater topo I inhibitory effect exhibited potent cytotoxicity. Piperazine-substituted derivative, 5b, with potent topo I and moderate topo II activities intercalated between DNA bases and interacted with topos through H-bonds at the DNA cleavage site of a docking model. Moreover, flow cytometry indicated that cytotoxic 3-heteroarylisoquinolinamines led to accumulation of human cervical (HeLa) cancer cells in the different phases of the cell cycle before apoptosis. Taken together, 3-heteroarylisoquinolinamines possessed potent cytotoxicity with topos and cell cycle inhibitory activities.