Characterization of telomeric repeats in metaphase chromosomes and interphase nuclei of Syrian Hamster Fibroblasts.

Background: Rodents have been reported to contain large arrays of interstitial telomeric sequences (TTAGGG)n (ITS) located in pericentromeric heterochromatin. The relative sizes of telomeric sequences at the ends of chromosomes (TS) and ITS in Syrian hamster (Mesocricetus auratus) cells have not been evaluated yet, as well as their structural organization in interphase nuclei.

Results: FISH signal distribution analysis was performed on DAPI-banded metaphase chromosomes of Syrian hamster fibroblasts, and relative lengths of telomere signals were estimated.

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues.

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications.

Chapter 6. Application of new methods for detection of DNA damage and repair.

New methods for detecting DNA damage and repair are reviewed and their potential significance is discussed. These include methods based on analysis of DNA damage-induced chromatin modifications, cytological detection of DNA repair synthesis, damage-induced immobilization of repair proteins and living cell imaging. Special attention is paid to current methods of detection of modifications of histones and other proteins associated with DNA double-strand breaks which represent most dangerous genome damage.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells.

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay).