Rabbit Anti-Human TOP1 Polyclonal antibody

The expression profiles of genes involved in DNA repair, namely MtTdp1 (tyrosyl-DNA phosphodiesterase), top1 (DNA topoisomerase I), MtTFIIS (transcription elongation factor II-S) and MtTFIIS-like, were evaluated in Medicago truncatula Gaertn. during seed imbibition carried out with the osmotic agent polyethylene glycol (PEG6000, 100 g/L). The use of PEG6000 resulted in delayed water up-take by seeds, and reduced levels of oxidative DNA damage, measured in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG) were observed compared to seeds imbibed with water. The prolonged exposure to PEG6000 caused an increase in DNA oxidative damage; after 24 h of treatment with the osmotic agent, the estimated amount of 8-oxo-dG was 1.25-fold higher compared to the value detected in seeds imbibed with water. Three days after imbibition, consistent cell damage and reactive oxygen species (ROS) production were also detected in radicles emerging from the PEG-treated seeds. All of the tested genes were known to be up-regulated during seed imbibition, with the highest transcript levels accumulating at approximately 8-12 h of rehydration. Exposure to PEG6000 caused a delayed up-regulation of MtTdp1 alpha and MtTdp1 beta genes, with transcript peaks occurring at 12-24 h, when the highest levels of DNA damage were also recorded. For the top I, MtTFIIS and MtTFIIS-like genes, different expression profiles were observed in response to PEG6000. The possible roles of these genes in the repair response activated during seed imbibition are discussed. (C) 2010 Elsevier GmbH. All rights reserved.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.