DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.

SummaryType II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes. Introduction DNA molecules in all cellular organisms are subjected to topological constraints. These constraints arise from the size and the bi-helical structure of the DNA. In the double helix, the two strands are topologically linked in vivo (as long as the phosphodiester backbone is intact) either by ring closure (in the case of circular DNA) or by the binding of macromolecular complexes. This topological structure prevents the free rotation of the two strands around each other, unless they can cross each other via transient breaks in the phosphodiester backbone. DNA topoisomerases (EC 5.99.1.3) appear to have evolved to solve this topological problem and participate in all DNA transactions that require partial or complete unwinding of the two DNA strands (replication, transcription, recombination, chromatin remodelling) (for reviews, see Refs.

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.

A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5-ends of the broken DNA. However, sequence analysis of the doublestrand breaks revealed that they are all characterized by staggered two-nucleotide long 5 overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5 extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.Type II DNA topoisomerases are ubiquitous enzymes that catalyze the ATP-dependent transport of one DNA duplex through a second DNA segment via a transient double-strand break (1). This ability to modulate the topological state of DNA is essential in major biological processes such as replication, recombination, and transcription (2). Until recently, these enzymes were thought to form a single family of homologous proteins. The discovery of DNA topoisomerase VI (topoVI) 1 in hyperthermophilic archaea has modified this classification. Type II DNA topoisomerases are now subdivided into two subfamilies, type IIA and IIB DNA topoisomerases (Fig. 1) (3).The type IIA subfamily contains three cellular representatives: eucaryotic DNA topoisomerase II (topoII), bacterial DNA gyrase, and DNA topoisomerase IV (topoIV). DNA gyrase and topoIV are heterotetramers composed of two subunits GyrA and GyrB, and ParC and ParE, respectively, while the eucaryotic enzyme is a homodimer. Despite this difference in quaternary structure, protein sequences comparison revealed that GyrB and ParE subunits are homologous to the N-terminal part of the eucaryotic enzyme while GyrA and ParC are homologous to the C-terminal half. These similarities were further confirmed by structural analysis of several fragments of Saccharomyces cerevisiae DNA topoII and Escherichia coli DNA gyrase (4 -7).Archaeal topoVI is the prototype of the recently described type IIB DNA topoisomerase subfamily (3). Thi...

BackgroundKabul (Afghanistan) is a major focus of cutaneous leishmaniasis (CL) caused by Leishmania tropica. Microscopy remains the reference test for diagnosis despite its low performance. We evaluated whether Loopamp™ Leishmania Detection Kit (Loopamp) and CL Detect™ Rapid Test (CL Detect), detecting Leishmania DNA and antigen, respectively could improve CL diagnosis.MethodsA diagnostic accuracy study with prospective inclusion was conducted in a leishmaniasis reference clinic in Kabul. Slit skin samples from CL suspects were analysed by microscopy. Samples taken with a dental broach were tested with CL Detect, Loopamp, and PCR. All samples were transferred to the Academic Medical Center (AMC, the Netherlands) for PCR and Loopamp analyses. The diagnostic performance of the tests was evaluated against a reference combining microscopy and PCR.Findings274 CL suspects were included in the study. In Kabul, CL Detect had a 65·4% sensitivity [95% Confidence Interval (CI): 59.2–71.2%] and a 100% specificity [95% CI: 80.5–100%], while these were 87.6% [95%CI: 82.9–91.3%] and 70.6% [95% CI: 44.0–89.7%] for Loopamp. At AMC the Loopamp's sensitivity (92.2% [95% CI: 88.2–95.2%]) and specificity (94.1% [95% CI: 71.3–99.8%]) were higher. An algorithm where CL Detect negative suspects would be tested by Loopamp yielded a 93.4% sensitivity [95% CI: 89.6–96.1%] and a 94.1% specificity [95% CI: 71.3–99.8%] when Loopamp's performance at AMC was used.InterpretationThe high specificity of CL Detect and the performance of Loopamp allow their use in a diagnostic algorithm that would minimize the number of CL patients referred for confirmation.FundFederal Ministry of Education and Research, Germany.

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One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

BackgroundInsecticide resistance seriously threatens the efficacy of vector control interventions in malaria endemic countries. In Afghanistan, the status of insecticide resistance is largely unknown while distribution of long-lasting insecticidal nets has intensified in recent years. The main objective of this study was thus to measure the level of resistance to four classes of insecticides in provinces with medium to high risk of malaria transmission.MethodsAdult female mosquitoes were reared from larvae successively collected in the provinces of Nangarhar, Kunar, Badakhshan, Ghazni and Laghman from August to October 2014. WHO insecticide susceptibility tests were performed with DDT (4 %), malathion (5 %), bendiocarb (0.1 %), permethrin (0.75 %) and deltamethrin (0.05 %). In addition, the presence of kdr mutations was investigated in deltamethrin resistant and susceptible Anopheles stephensi mosquitoes collected in the eastern provinces of Nangarhar and Kunar.ResultsAnalyses of mortality rates revealed emerging resistance against all four classes of insecticides in the provinces located east and south of the Hindu Kush mountain range. Resistance is observed in both An. stephensi and Anopheles culicifacies, the two dominant malaria vectors in these provinces. Anopheles superpictus in the northern province of Badakhshan shows a different pattern of susceptibility with suspected resistance observed only for deltamethrin and bendiocarb. Genotype analysis of knock down resistance (kdr) mutations at the voltage-gated channel gene from An. stephensi mosquitoes shows the presence of the known resistant alleles L1014S and L1014F. However, a significant fraction of deltamethrin-resistant mosquitoes were homozygous for the 1014L wild type allele indicating that other mechanisms must be considered to account for the observed pyrethroid resistance.ConclusionsThis study confirms the importance of monitoring insecticide resistance for the development of an integrated vector management in Afghanistan. The validation of the kdr genotyping PCR assay applied to An. stephensi collected in Afghanistan paves the way for further studies into the mechanisms of insecticide resistance of malaria vectors in this region.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1149-1) contains supplementary material, which is available to authorized users.