Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase IIα

AbstractBisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme’s Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme

New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability

Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction

OXIDATION OF N-(4-CHLOROPHENYL)-N'-HYDROXYGUANIDINE TO N-(4-CHLOROPHENYL) UREA AND NITRIC OXIDE WITH PHOTOEXCITED IRON PORPHYRINS

Photochemical excitation (l> 350 nm) of chloro[meso-tetrakis(2,6-dichloroporphyrin]iron (III) Fe(III)(TDCPP)Cl] and chloro[meso-tetra(a,a,a,a-pivalamidophenyl)porphyrin]-iron (III) [Fe(III)(Tpiv)PPCl] induces the oxidation of coordinated N-(4-chlorophenyl)-N'-hydroxyguanidine (1) by molecular oxygen, to give iminoxyl radicals and the Fe(II)O2 adduct. This complex can be accumulated in significant amounts using [Fe(III)(TpivPP)Cl]. The primary photoproducts give rise to secondary reactions that lead to the formation of N-(4-chlorophenyl)urea (2) as the main end product of 1. The conversion of 1 into 2 is accompanied by the formation of NO, as revealed both by an ESR spin trapping technique and in the form of its stable end-products NO2- and NO3-. The presence of 1-methyl imidazole coordinated in axial position has a significant positive effect on the photoinduced production of 2 and NO from the hydroxyguanidine 1. The observation that radical scavengers inhibit the photooxidation process strongly supports the possibility that the reaction pathway resembles a radical-type autooxidation mechanism, where the very fast reaction of O2 with the ferrous porphyrin in the presence of the photogenerated iminoxyl radical should yield an iron-peroxo intermediate as precursor of the urea 2 and NO