End-processing during non-homologous end-joining: a role for exonuclease 1

Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5′-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1

Distinct requirements for the Rad32(Mre¹¹) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA

For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32Mre11 nuclease activity is involved in the removal of both Top2 from 5′ DNA ends as well as Top1 from 3′ ends in vivo. A ctp1CtIP deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1CtIP plays distinct roles in removing topoisomerases from 5′ and 3′ DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs

Effects of DNA and protein size on substrate cleavage by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the hydrolysis of phosphodiester linkages between a DNA 3′ phosphate and a tyrosine residue as well as a variety of other DNA 3′ substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nucleotides indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has previously been found that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid long topoisomerase I-derived peptide were efficiently cleaved by TDP1, but like the full length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo

STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G<inf>2</inf> Arrest in the Absence of DNA Damage

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent. © 2013 Kim et al

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

Human topoisomerase IIα uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen

The dual-acting chemotherapeutic agent Alchemix induces cell death independently of ATM and p53

YesTopoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes

Evaluation of Two Models for Human Topoisomerase I Interaction with dsDNA and Camptothecin Derivatives

Human topoisomerase I (Top1) relaxes supercoiled DNA during cell division. Camptothecin stabilizes Top1/dsDNA covalent complexes which ultimately results in cell death, and this makes Top1 an anti-cancer target. There are two current models for how camptothecin and derivatives bind to Top1/dsDNA covalent complexes (Staker, et al., 2002, Proc Natl Acad Sci USA 99: 15387–15392; and Laco, et al., 2004, Bioorg Med Chem 12: 5225–5235). The interaction energies between bound camptothecin, and derivatives, and Top1/dsDNA in the two models were calculated. The published structure-activity-relationships for camptothecin and derivatives correlated with the interaction energies for camptothecin and derivatives in the Laco et al. model, however, this was not the case for several camptothecin derivatives in the Stacker et al. model. By defining the binding orientation of camptothecin and derivatives in the Top1/dsDNA active-site these results allow for the rational design of potentially more efficacious camptothecin derivatives

Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

<p>Abstract</p> <p>Background</p> <p>DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM).</p> <p>Results</p> <p>We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed proteins that are highly likely to be involved in the repair process. A new web service, INTREPED, has been made available for the immediate search and annotation of DNA repair proteins in newly sequenced genomes.</p> <p>Conclusion</p> <p>Despite complexity due to a multitude of repair pathways, combinations of sequence, structure, and homology with Support Vector Machines offer good methods in addition to existing homology searches for DNA repair protein identification and functional annotation. Most importantly, this study has uncovered relationships between the size of a genome and a genome's available repair repetoire, and offers a number of new predictions as well as a prediction service, both which reduce the search time and cost for novel repair genes and proteins.</p

Structural Basis of Gate-DNA Breakage and Resealing by Type II Topoisomerases

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg2+ ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics