DNA Processing Reactions Research Articles

Pharmacological properties of dicyanidoaurate(I)-based complexes: characterization and single crystal X-ray analysis

AbsractThe synthesis of three bimetallic cyanido complexes with edbea [2,2′-(ethylenedioxy)bis(ethylamine)] ligand is reported. [NiII(μ-edbea)2{Au(μ-CN)2}2]n (1), [{CuII(edbea)}2{Au(μ-CN)2}4]n (2) and [CdII(edbea)2][Au(CN)2]2·H2O (3) were fully characterized by elemental, infrared, XRD (3), ESI-MS and thermal analysis. The DNA/BSA binding properties of these complexes were evaluated by spectrophotometric titration, fluorometric ethidium bromide kinetics, and DNA electrophoresis studies and their partially minor groove binding mode between the base pairs of DNA and electrostatic interaction between the amino acid residues of BSA were explained. The complexes were tested for their pharmacological properties. These molecules had excellent in vitro antiproliferative activity and also exhibited a strong tumor inhibiting effect against HT29, HeLa, C6 and Vero cell lines. These complexes had metastatic features as they are able to reduce cell migration activity and suppress tumor growth in vitro. Analysis of the DNA topoisomerase I relaxing activity indicates that the complexes do not inhibit topoisomerase I which regulates the topological states of the DNA double helix during DNA processing reactions. The TUNEL and DNA laddering assay results indicated that these compounds may destroy cell maintenance by triggering apoptosis. Immunohistochemistry staining analysis demonstrated that these complexes significantly decreased the expression of Bcl-2 in HeLa and HT29 cells while increasing the expression of P53 levels. Overall, the potent antiproliferative activity, low cytotoxic effect, good solubility, and micro molar range dosage observed for these complexes emphasizes their potential as anticancer drug candidates.

Assembling the Human Resectosome on DNA Curtains.

DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome-a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom's helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.

Electronic pH switching of DNA triplex reactions

Remote electronic control of fast DNA processing reactions such as S–S-ligation is achievedviapH switching of triplex structures.

Domain Architecture of RecQ Helicase Defines Mechanochemical Linkage via Multipartite Interactions with DNA Substrate during Unwinding Activity

RecQ helicases are ubiquitous enzymes that safeguard the genome by playing multiple roles in DNA repair, replication and recombination. They are unique in their capability to process a wide range of non-canonical structures associated with DNA metabolic intermediates. DNA-binding domains linked to the RecQ helicase core, including the winged-helix (WHD) and helicase-and-RnaseD-C-terminal (HRDC) domains, are thought to confer substrate specificity and modulate enzymatic activities. We combined ensemble biophysical and single-molecule magnetic tweezers assays to determine the mechanochemical linkage between ATP hydrolytic and DNA-restructuring activities of E. coli RecQ constructs of varying domain architecture. We compared the activities of wild-type, HRDC point mutant and deletion mutant, and WHD-HRDC deletion mutant constructs using several defined DNA structures and experimental geometries. We show that the WHD enhances unwinding processivity by stabilizing enzyme-DNA interactions, whereas the HRDC domain increases the overall DNA affinity but hinders the unwinding and ATPase activities of the helicase through interactions with single-stranded (ss) DNA regions. Intriguingly, HRDC-ssDNA interactions stabilize the pausing of the helicase during unwinding of a DNA hairpin in which both nascent ssDNA strands are mechanically strained, but not that of gapped duplex DNA in which one of the liberated ssDNA strands is mechanically unconstrained. Our study reveals that both the DNA substrate geometry and the contribution of auxiliary DNA-binding domains greatly influence the mechanochemical behavior of the helicase and the outcome of DNA-processing reactions, bearing consequences on the diverse in vivo actions of RecQ enzymes.

Rescue of Stalled Replication Forks by the DNA Translocase FANCM

Fanconi anemia (FA) is a chromosomal instability syndrome that manifests with congenital abnormalities, bone marrow failure and cancer predisposition. To date, fourteen FA genes have been identified and implicated in a ubiquitin signaling pathway, in S phase checkpoint activation, in lesion bypass and in DNA double‐strand break (DSB) repair by homologous recombination.FANCM is a DNA translocase that acts as a molecular scaffold involved in multiple DNA damage signaling and processing reactions. FANCM interacts with : 1) the checkpoint protein HCLK2 to facilitate DNA damage signaling; 2) with the Bloom's complex at stalled forks; 3) and with the FA ubiquitin ligase complex, promoting its recruitment to chromatin and the monoubiquitination of FANCD2 and FANCI.Our biochemical studies revealed that FANCM binds and translocates the branch point of DNA structures that mimic replication and repair intermediates, suggesting that FANCM may remodel replication intermediates to promote recovery. Using a molecular combing approach, we have observed that FANCM facilitates the restart of replication forks stalled by DNA topoisomerase I inhibitors. Our recent efforts have focused on studying the role new DNA damage response proteins that interact with FANCM.Research support: ARC Association pour la Recherche sur le Cancer, ANR Agence Nationale de la Recherche, SNSF Swiss National Science Foundation.

Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3'-S-phosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.

The F plasmid‐encoded TraM protein stimulates relaxosome‐mediated cleavage at oriT through an interaction with TraI

Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer - they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5'-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.

Functional coupling between the two active sites during Tn10 transposition buffers the mutation of sequences critical for DNA hairpin processing

DNA processing reactions often involve multiple components acting in concert to achieve the desired outcome. However, it is usually difficult to know how the components communicate and cooperate to orchestrate an ordered series of events. We address this question in the context of the Tn 10 transposition reaction, in which the DNA cleavage and joining events occur within a higher-order complex containing a transposase dimer, two transposon ends and the DNA-bending host-factor IHF (Integration Host Factor). Previously it was shown that the complex is asymmetric. The a side consists of an IHF protomer initially immobilized by a DNA-loop, but subsequently used to promote conformational changes required for the cleavage steps. The beta side of the complex was considered to fulfil a more passive role. Here we show that the a side of the complex promotes coupled conformational changes at both transposon ends, while the a and beta sides communicate and cooperate to dominate different phases of the transposition reaction. Together, these effects provide for a robust response to critical changes in the transposon end. These findings also explain the intriguing genetic phenotypes of a series of previously reported Tn10 mutants and have consequences for the evolution of new elements.

Genome-wide expression profile of the mnn2Δ mutant of Saccharomyces cerevisiae

The MNN2 gene of S. cerevisiae encodes an alpha (1,2) mannosyl transferase required for branching the outer chain of N-linked oligosaccharides (Rayner J.C. and Munro S. 1998. J. Biol. Chem. 273: 26836-26843) and it also seems to have some effect on the transfer of mannosyl phosphate groups to the inner core (Olivero I. et al. 2000. FEBS Lett. 475: 111-116). In order to reveal possible interactions of MNN2 expression with other cellular pathways, we analyzed the transcriptome of the deletion mutant S. cerevisiae mnn2 Delta using cDNA microarrays. We found 151 genes that showed an altered expression level of > or =2-fold, 58 of them up-regulated and 93 down-regulated. Quite a high proportion of these genes (29%) encode unclassified proteins. In contrast to other defects affecting the integrity of the cell wall, deletion of MNN2 does not stimulate the expression of any of the genes included in the previously defined 'cell wall compensatory cluster' (Lagorce et al. 2003. J. Biol. Chem. 278: 20345-20357). We also found that 15% of the selected genes are related to central metabolic pathways. In addition, the mnn2 Delta strain seems to have a certain level of stimulation of DNA processing reactions while some genes involved in intracellular transport pathways are under-regulated.

“In‐gel patch electrophoresis:” A new method for environmental DNA purification

Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications.

Covalent trapping of protein-DNA complexes.

High-resolution structural studies of protein-DNA complexes have proven to be an invaluable means of understanding the diverse functions of proteins that manage the genome. Most of the structures determined to date represent proteins bound noncovalently to various DNA sequences or structures. Although noncovalent complexation is often adequate to study the structures of proteins that have robust, specific interactions with DNA, it is poorly suited to the study of transient intermediates in enzyme-catalyzed DNA processing reactions or of complexes that exist in multiple equilibrating forms. In recent years, strategies developed for the covalent trapping of protein-DNA complexes have begun to show promise as a window into an otherwise inaccessible world of structure.

The RAG1/RAG2 Complex Constitutes a 3′ Flap Endonuclease: Implications for Junctional Diversity in V(D)J and Transpositional Recombination

During V(D)J recombination, processing of branched coding end intermediates is essential for generating junctional diversity. Here, we report that the RAG1/RAG2 recombinase is a 3′ flap endonuclease. Substrates of this nuclease activity include various coding end intermediates, suggesting a direct role for RAG1/RAG2 in generating junctional diversity during V(D)J recombination. Evidence is also provided indicating that site-specific RSS nicking involves RAG1/RAG2-mediated processing of a localized flap-like structure, implying 3′ flap nicking in multiple DNA processing reactions. We have also demonstrated that the bacterial transposase Tn10 contains a 3′ flap endonuclease activity, suggesting a mechanistic parallel between RAG1/RAG2 and other transposases. Based on these data, we propose that numerous transposases may facilitate genomic evolution by removing single-stranded extensions during the processing of excision site junctions.

IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes.

Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF- and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF- strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding.

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products.

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.

Multiple DNA Processing Reactions Underlie Tn 7Transposition

The bacterial transposon Tn 7uses a cut and paste mechanism to translocate between non-homologous insertion sites. In the first step of recombination, double-strand breaks at each transposon end disconnect the element from the donor backbone; in the second step, the now exposed 3′ transposon ends join to the target DNA. To dissect the chemical steps in these reactions, we have used mutant transposons altered at and near their extreme termini. We find that the initiating double-strand breaks result from a collaboration of two distinct DNA strand processing activities, one mediating cleavages at the 3′ ends of Tn 7, which can be blocked by changes at the transposon tips, and another mediating cleavages at the 5′ ends. The joining of exposed 3′ transposon ends to the target DNA can be blocked by changing the transposon tips. Our results suggest that the target joining step occurs through two usually concerted, but actually separable, reactions in which individual 3′ transposon ends are joined to separate strands of the target DNA. Thus Tn 7transposition involves several distinct DNA processing reactions: strand cleavage and strand transfer reactions at the 3′ ends of the transposon, and separate strand cleavage reactions at the 5′ ends of the transposon.

Localized denaturation of oriT DNA within relaxosomes of the broad-host-range plasmid R1162.

The broad-host-range, multicopy plasmid R1162 is efficiently mobilized during conjugation by the self-transmissible plasmid R751. The relaxosome, a complex of plasmid DNA and R1162-encoded proteins, forms at the origin of transfer (oriT) and is required for mobilization. Transfer is initiated by strand- and site-specific nicking of the DNA within this structure. We show by probing with potassium permanganate that oriT DNA is locally melted within the relaxosome, in the region from the inverted repeat to the site that is nicked. Mutations in this region of oriT, and in genes encoding the protein components of the relaxosome, affect both nicking and melting of the DNA. The nicking protein in the relaxosome is MobA, which also ligates the transferred linear, single strand at the termination of a round of transfer. We propose that there is an underlying similarity in the substrates for these two MobA-dependent, DNA-processing reactions. We also show that MobA has an additional role in transfer, beyond the nicking and resealing of oriT DNA.

DNA processing reactions in bacterial conjugation.

Bacterial conjugation is an important source of genetic plasticity. The initiation complex for conjugative transfer of transmissible plasmids--the relaxosome--is a specific DNA-protein structure that has been isolated from cells and reconstituted from purified components in vitro. Complexes containing uncleaved DNA and DNA cleaved at the nicsite in the origin of transfer (oriT) coexist in equilibrium. Relaxase is usually loaded onto oriT by accessory DNA-binding proteins. Relaxase catalyzes cleavage of a specific phosphodiester bond at nic and becomes covalently linked through a tyrosyl residue to the 5' terminus of the cleaved strand. Cleaved DNA may be unwound for transfer by a plasmid-encoded helicase. Single-strand transfer is thought to occur by a replicative rolling circle mechanism. Termination of a round of transfer is achieved by the cleaving-joining activity of the relaxase linked to the 5' end of the transferring strand. Relationships between DNA processing reactions and conjugative interactions of cell envelopes are particularly obscure aspects of the conjugation cycle.

DNA Processing Reactions in Bacterial Conjugation

Bacterial conjugation is an important source of genetic plasticity. The initiation complex for conjugative transfer of transmissible plasmids—the relaxosome—is a specific DNA-protein structure that has been isolated from cells and reconstituted from purified components in vitro. Complexes containing uncleaved DNA and DNA cleaved at the nicsite in the origin of transfer (oriT) coexist in equilibrium. Relaxase is usually loaded onto oriT by accessory DNA-binding proteins. Relaxase catalyzes cleavage of a specific phosphodiester bond at nic and becomes covalently linked through a tyrosyl residue to the 5′ terminus of the cleaved strand. Cleaved DNA may be unwound for transfer by a plasmid-encoded helicase. Single-strand transfer is thought to occur by a replicative rolling circle mechanism. Termination of a round of transfer is achieved by the cleaving-joining activity of the relaxase linked to the 5′ end of the transferring strand. Relationships between DNA processing reactions and conjugative interactions of cell envelopes are particularly obscure aspects of the conjugation cycle.

V(D)J recombination in ataxia telangiectasia, Bloom's syndrome, and a DNA ligase I-associated immunodeficiency disorder.

Ataxia telangiectasia (AT) and Bloom's syndrome (BS) patients are characterized by sensitivity to radiation, increased lymphoid malignancy, and frequent translocations to the antigen receptor loci. Because of these features, there has been a persistent question as to whether the V(D)J recombinase might be abnormal in cells from these patients. Such abnormalities might be due to inappropriate to inaccurate expression of components of the V(D)J recombinase or due to mutation in a component shared between V(D)J recombination and other cellular processes, such as DNA repair. Bloom's syndrome is associated with a ligation deficiency, and this activity may contribute in the end resolution steps of both site-specific and general DNA-processing reactions. In the current study, we have activated V(D)J recombination in normal, AT, and BS fibroblasts and in fibroblasts from a patient with mutations that largely abolish DNA ligase I activity. We find that the signal and coding joint formation of the V(D)J recombination reaction are entirely normal in AT, BS, and DNA ligase I mutant cells. In addition to ruling out abnormalities of the V(D)J recombinase in AT, BS, and DNA ligase I mutant cells, these studies suggest that DNA ligase I is unlikely to be required for signal or coding end joining in the V(D)J recombination reaction.