Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds.

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.

Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts.

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

Abortive activity of Topoisomerase I: a challenge for genome integrity?

Single-strand breaks (SSB) are discontinuities in one strand of the DNA double helix and are the most common type of damages that arise in cells. SSBs arise mainly from direct attack by intracellular metabolites, however, also essential nuclear processes generate SSBs as intermediates. During the catalytic cycle of DNA topoisomerase I (Top1) a SSB is generated, which is normally transient and rapidly resealed by the enzyme. However, several situations can stabilize a Top1-generated SSB, and this poses the risk of converting the SSB into a double strand break (DSB) if encountered by the replication machinery. A DSB is a more serious treat for cells as it can fuel chromosomal rearrangements and thus jeopardize genome stability and cause cells to become cancerous. In this perspective, we discuss the cellular consequences of Top1-generated damage during DNA replication with focus on the differences between endogenous Top1-generated damage and Top1 damage generated due to the use of the drug camptothecin.

Minimal Resection Takes Place during Break-Induced Replication Repair of Collapsed Replication Forks and Is Controlled by Strand Invasion.

A natural and frequently occurring replication problem is generated by the action of topoisomerase I (Top1). Trapping of Top1 in a cleavage complex on the DNA generates a protein-linked DNA nick (PDN), which upon DNA replication can be transformed into a one-ended double-strand break (DSB). Break-induced replication (BIR) has been recognized as a critical repair mechanism of one-ended DSBs. Here, we have investigated resection at a one-ended DSB formed exclusively during replication due to Top1-mimicking damage. We show that resection is minimal, and only when strand invasion is abolished is extensive resection detected. When DNA synthesis is slowed by hydroxyurea treatment, extended resection is not observed, which suggests that strand invasion and/or heteroduplex formation restrains resection. Our results demonstrate that the BIR pathway acting during S phase is tailored to prevent hazardous effects of naturally and frequently occurring DNA breaks such as Top1-generated PDNs.

Using the Flp Recombinase to Induce Site-Specific Protein-DNA Nicks.

A natural and frequent occurring replication insult is generated by the action of DNA Topoisomerase I (Top1). When Top1 gets trapped in a cleavage complex on the DNA, a protein-linked DNA nick (PDN) is generated. Today it is known that PDNs are generated at a high incidence in the cell. If not rapidly removed, PDNs can have a profound impact on cell destiny, as a nick in proliferating cells is passively transformed into a single-ended DSB, when encountered by the replication machinery. A DSB can in turn lead to chromosomal rearrangements and thus jeopardize genome stability if not appropriately repaired. In order to study repair pathways associated with PDNs, we have developed a cellular system (Flp-nick), where we can generate a single PDN at a specific genomic site in the model organism Saccharomyces cerevisiae. The system takes advantages of the Flp recombinase, which catalytically operates like Top1 by generating a nick in the DNA backbone and during this process becomes covalently linked to the DNA. Flp cleaves at well-defined target sites. Thus, a target site has been inserted in the genome and a mutant Flp, which cleaves but do not religate, is expressed. In this way, a single PDN mimicking the one generated by Top1 is induced at a known genomic site. The Flp-nick system allows detailed molecular analysis of repair pathways associated with this type of damage and can be designed to study repair at any genomic context.

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination.

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.

Most spontaneous DNA double-strand breaks (DSBs) result from replication-fork breakage. Break-induced replication (BIR), a genome rearrangement-prone repair mechanism that requires the Pol32/POLD3 subunit of eukaryotic DNA Polδ, was proposed to repair broken forks, but how genome destabilization is avoided was unknown. We show that broken fork repair initially uses error-prone Pol32-dependent synthesis, but that mutagenic synthesis is limited to within a few kilobases from the break by Mus81 endonuclease and a converging fork. Mus81 suppresses template switches between both homologous sequences and diverged human Alu repetitive elements, highlighting its importance for stability of highly repetitive genomes. We propose that lack of a timely converging fork or Mus81 may propel genome instability observed in cancer.

DNA Topoisomerases Are Required for Preinitiation Complex Assembly during GAL Gene Activation.

To investigate the importance of topoisomerases for transcription of the galactose induced genes, we have studied the expression of GAL1, GAL2, GAL7 and GAL10 in Saccharomyces cerevisiae cells deficient for topoisomerases I and II. We find that topoisomerases are required for transcriptional activation of the GAL genes, but are dispensable for ongoing transcription, eliminating a role of the enzymes in transcriptional elongation. Furthermore, we demonstrate that promoter chromatin remodeling of the GAL genes is unaffected in the topoisomerase deficient strain. However, the cells fail to successfully recruit RNA polymerase II due to an inability of the TATA-binding protein (TBP) to bind to the TATA box in these promoters. We therefore argue that topoisomerases are required for accurate assembly of the preinitiation complex at the promoters of the GAL genes.

Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations.

Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.

A Rad53 independent function of Rad9 becomes crucial for genome maintenance in the absence of the Recq helicase Sgs1.

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

MRX protects fork integrity at protein-DNA barriers, and its absence causes checkpoint activation dependent on chromatin context.

To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein-DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein-DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.

Studying repair of a single protein-bound nick in vivo using the Flp-nick system.

The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target (FRT) site, which has been integrated into the yeast genome. Upon cleavage at the FRT site, the Flp mutant becomes covalently linked to the 3' DNA end at the nick in an irreversible manner, as the mutant fails to accomplish the required religation process. Thus, the established damage mimics a stabilized topoisomerase I-DNA cleavage complex. DNA topoisomerases are ubiquitous enzymes that relieve topological stress in the DNA arising during DNA replication or transcription. During this process, they make transient enzyme-DNA cleavage complexes, which normally are reversed by a rapid ligation step. However, aberrant long-lived enzyme-DNA complexes may occur frequently due to either endogenous or exogenous damage, and the cellular repair machinery therefore needs to be able to eliminate this type of damage. This chapter describes how to establish the Flp-nick system in S. cerevisiae, how to detect FlpH305L at the FRT site using a modified chromatin immunoprecipitation assay where formaldehyde fixation is omitted, and how to monitor nicking at the FRT site by alkaline denaturing gel analysis.

DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

A Flp-nick system to study repair of a single protein-bound nick in vivo.

We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I-DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I-DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.

RecQ helicases: lessons from model organisms.

RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication, and that proteins involved in the S-phase checkpoint are a principal defence against such instability. Cells that lack functional RecQ helicases exhibit phenotypes consistent with an inability to fully resume replication fork progress after encountering DNA damage or fork arrest. In this review we will concentrate on the various functions of RecQ helicases during S phase in model organisms.

Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1Delta with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1Delta mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1Delta mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1Delta cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance.

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.

Hindering the strand passage reaction of human topoisomerase IIalpha without disturbing DNA cleavage, ATP hydrolysis, or the operation of the N-terminal clamp.

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

The transducer domain is important for clamp operation in human DNA topoisomerase IIalpha.

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase II alpha enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.

RecQ helicases and topoisomerase III in cancer and aging.

RecQ helicases have in recent years attracted increasing attention due to the important roles they play in maintaining genomic integrity, which is essential for the life of a cell and the survival of a species. Humans with mutations in RecQ homologues are cancer prone and suffer from premature aging. A great effort has therefore been made to understand the molecular mechanisms and the biological pathways, in which RecQ helicases are involved. It has become clear that these enzymes work in close concert with DNA topoisomerase III, and studies in both yeast and mammalian systems point to a role of the proteins in processes involving homologous recombination. In this review we discuss the genetic and biochemical evidence for possible functions of RecQ helicases and DNA topoisomerase III in multiple cellular processes such as DNA recombination, DNA replication, and cell cycle checkpoint control.