Low Level Phosphorylation of Histone H2AX on Serine 139 (gammaH2AX) Is Not Associated with DNA Double-Strand Breaks

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities

Quantitative analysis of DNA damage induced by DNA topoisomerase I and II inhibitors

Inhibitory topoizomeraz typu I i II są jedną z wielu klas substancji wykorzystywanych w terapii przeciwnowotworowej. Topoizomerazy umożliwiają relaksację superskrętów DNA przez odwracalne przerwanie nici DNA w reakcjach transestryfikacji. Działanie inhibitorów topoizomeraz polega na uniemożliwieniu odtworzenia wiązania fosfodiestrowego i w konsekwencji zatrzymaniu pracy enzymu. Może to prowadzić do powstania uszkodzeń DNA. Topoizomery uczestniczą m.in. w replikacji, transkrypcji i segregacji chromosomów. Nie jest określony udział każdego z typów topoizomeraz w procesach zachodzących w fazie S cyklu komórkowego.Celem pracy było zbadanie lokalizacji miejsc wykrycia uszkodzeń chromatyny i lokalizacji miejsc naprawy uszkodzeń DNA na drodze homologicznej rekombinacji wywołanych inhibitorami topoizomeraz typu I i II oraz określenie zależności liczbowej i przestrzennej tych miejsc odpowiedzi komórkowej na uszkodzenia względem miejsc replikacji na różnych etapach fazy S cyklu komórkowego. W tym celu wykorzystano barwienie immunofluorescencyjne, metodę detekcji syntezy DNA w reakcji typu „click-iT” i laserową skaningową mikroskopię konfokalną.Pokazano, że pod wpływem działania mitoksantronu (inhibitora topoizomeraz typu II) występuje wyższy poziom uszkodzeń chromatyny niż w przypadku zastosowania kamptotecyny (inhibitora topoizomerazy typu I) lub etopozydu (inhibitora topoizomerazy typu II), który nie jest skorelowany z wyższą liczbą miejsc naprawy na drodze homologicznej rekombinacji. Ponadto na podstawie relacji przestrzennej miejsc wykrycia uszkodzenia i miejsc replikacji wyciągnięto wniosek, że chromatyna jest najmniej wrażliwa na uszkodzenia miejsc replikacji wywołane etopozydem i kamptotecyną na początku fazy S.DNA topoisomerase I and II inhibitors are one of the many classes of drugs used in cancer therapy. Topoisomerases relax DNA supercoiling by transient cleavage of DNA strand in transestrification reactions. The mechanism of action of topoisomerase I and II inhibitors preclude the enzyme from re-ligation of the phosphodiester bond in DNA, subsequently suspending enzyme activity. The action may induce DNA damage. DNA topoisomerase has functions in DNA replication, transcription and chromosome segregation. The extend to which each type of DNA topoisomerases participates in the processes in S-phase of the cell cycle.The aim of this study was to examine localization of the detection sites of DNA damage and localization of the sites of DNA damage repair by homologous recombination induced by DNA topoisomerase I and II inhibitors. The study aims also at defining the spacial and quantitive relationship between these sites and replication foci in subphases of S-phase of cell cycle. To achieve this goal immunofluorescence staining, detection of DNA synthesis in „click” reaction and laser confocal scanning microscopy was used.It has been demonstrated that mitoxantrone (topoisomerase II inhibitor) induces an increased level of detection of DNA damage comparing to camptothecin (topoisomerase I inhibitor) and etoposide (topoisomerase II inhibitor), which is not correlated with an increased number of sites of DNA damage repair by homologous recombination. In addition, examining distances in 3D space between DNA damage and replication foci it has been concluded that chromatin is the least sensitive to damage induced by etoposide and camptothecin in the early S phase of cell cycle

Activation of new replication foci under conditions of replication stress

<p>DNA damage, binding of drugs to DNA or a shortage of nucleotides can decrease the rate or completely halt the progress of replication forks. Although the global rate of replication decreases, mammalian cells can respond to replication stress by activating new replication origins. We demonstrate that a moderate level of stress induced by inhibitors of topoisomerase I, commencing in early, mid or late S-phase, induces activation of new sites of replication located within or in the immediate vicinity of the original replication factories; only in early S some of these new sites are also activated at a distance greater than 300 nm. Under high stress levels very few new replication sites are activated; such sites are located within the original replication regions. There is a large variation in cellular response to stress – while in some cells the number of replication sites increases even threefold, it decreases almost twofold in other cells. Replication stress results in a loss of PCNA from replication factories and a twofold increase in nuclear volume. These observations suggest that activation of new replication origins from the pool of dormant origins within replication cluster under conditions of mild stress is generally restricted to the original replication clusters (factories) active at a time of stress initiation, while activation of distant origins and new replication factories is suppressed.</p

Highly amyloidogenic two-chain peptide fragments are released upon partial digestion of insulin with pepsin

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein