Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA

Chemistry of the 8‐Nitroguanine DNA Lesion: Reactivity, Labelling and Repair

The 8-nitroguanine lesion in DNA is increasingly associated with inflammation-related carcinogenesis, whereas the same modification on guanosine 3',5'-cyclic monophosphate generates a second messenger in NO-mediated signal transduction. Very little is known about the chemistry of 8-nitroguanine nucleotides, despite the fact that their biological effects are closely linked to their chemical properties. To this end, a selection of chemical reactions have been performed on 8-nitroguanine nucleosides and oligodeoxynucleotides. Reactions with alkylating reagents reveal how the 8-nitro substituent affects the reactivity of the purine ring, by significantly decreasing the reactivity of the N2 position, whilst the relative reactivity at N1 appears to be enhanced. Interestingly, the displacement of the nitro group with thiols results in an efficient and specific method of labelling this lesion and is demonstrated in oligodeoxynucleotides. Additionally, the repair of this lesion is also shown to be a chemically feasible reaction through a reductive denitration with a hydride source

Antitumour and antimalarial activity of artemisinin–acridine hybrids

Artemisinin–acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2–4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle

Structure-based design of nucleoside-derived analogues as sulfotransferase inhibitors

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs

A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3′–5′ and 5′–3′ strands by rotating a single active site

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA

What are the factors that determine the types of questions that people commonly think make for a good question for scientists to answer?

The present online study investigates the properties of question that lay people agree on that makes a research question easy to understand, as well as worth answering. As yet there is limited understanding about the features of a question that in combination lead to an overall judgment that a question is good or not good. Graesser, Person, and Huber (1992) have proposed a GPH scheme that outlines two general features of questions that enable an overall assessment that a question is presented in a manner that makes it of value: “content” the structure of the information sought, “question-generation mechanism” the psychological processes––goals, plans, and knowledge––which bring about a question. The GPH Scheme lists also lists four specific properties that consider the question-generation mechanisms: 1) reducing, or correcting, a knowledge deficit; 2) monitoring common ground; 3) social coordination of action; and 4) control of conversation and attention. Up until now, there is no analysis of research questions based on the GPH scheme, and so the overall objective is to present participants with an online survey that is comprised of four sets of judgment probes that invite participants to consider a research question from the view of the following four properties of a question: 1) reducing, or correcting, a knowledge deficit; 2) monitoring common ground; 3) social coordination of action; and 4) control of conversation and attention. After they are presented with judgment probes related to each of these four properties, they will be invited to make an overall judgment as to whether they think the research question is worth answering, and the extent to which the research question is deemed a “good” question

What are the factors that determine the types of questions that people commonly think make for a good question for scientists to answer?

The present online study investigates the properties of question that lay people agree on that makes a research question easy to understand, as well as worth answering. As yet there is limited understanding about the features of a question that in combination lead to an overall judgment that a question is good or not good. Graesser, Person, and Huber (1992) have proposed a GPH scheme that outlines two general features of questions that enable an overall assessment that a question is presented in a manner that makes it of value: “content” the structure of the information sought, “question-generation mechanism” the psychological processes––goals, plans, and knowledge––which bring about a question. The GPH Scheme lists also lists four specific properties that consider the question-generation mechanisms: 1) reducing, or correcting, a knowledge deficit; 2) monitoring common ground; 3) social coordination of action; and 4) control of conversation and attention. Up until now, there is no analysis of research questions based on the GPH scheme, and so the overall objective is to present participants with an online survey that is comprised of four sets of judgment probes that invite participants to consider a research question from the view of the following four properties of a question: 1) reducing, or correcting, a knowledge deficit; 2) monitoring common ground; 3) social coordination of action; and 4) control of conversation and attention. After they are presented with judgment probes related to each of these four properties, they will be invited to make an overall judgment as to whether they think the research question is worth answering, and the extent to which the research question is deemed a “good” question