An ultrasoft X-ray multi-microbeam irradiation system for studies of DNA damage responses by fixed- and live-cell fluorescence microscopy

Localized induction of DNA damage is a valuable tool for studying cellular DNA damage responses. In recent decades, methods have been developed to generate DNA damage using radiation of various types, including photons and charged particles. Here we describe a simple ultrasoft X-ray multi-microbeam system for high dose-rate, localized induction of DNA strand breaks in cells at spatially and geometrically adjustable sites. Our system can be combined with fixed- and live-cell microscopy to study responses of cells to DNA damage

Global Analysis of Extracytoplasmic Stress Signaling in Escherichia coli

The Bae, Cpx, Psp, Rcs, and σE pathways constitute the Escherichia coli signaling systems that detect and respond to alterations of the bacterial envelope. Contributions of these systems to stress response have previously been examined individually; however, the possible interconnections between these pathways are unknown. Here we investigate the dynamics between the five stress response pathways by determining the specificities of each system with respect to signal-inducing conditions, and monitoring global transcriptional changes in response to transient overexpression of each of the effectors. Our studies show that different extracytoplasmic stress conditions elicit a combined response of these pathways. Involvement of the five pathways in the various tested stress conditions is explained by our unexpected finding that transcriptional responses induced by the individual systems show little overlap. The extracytoplasmic stress signaling pathways in E. coli thus regulate mainly complementary functions whose discrete contributions are integrated to mount the full adaptive response

Creating localized DNA double-strand breaks with microirradiation.

We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2\u27-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 μM BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.without figure

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNA-damaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of ≈30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatin-associated protein complex in living cells