Biomedical subjects

Robert Shroff

Publications and source records attributed to Robert Shroff.

3 recordsLinked to original sources

PubMed2006

Techniques for gamma-H2AX detection.

When a double-strand break (DSB) forms in DNA, many molecules of histone H2AX present in the chromatin flanking the break site are rapidly phosphorylated. The phosphorylated derivative of H2AX is named gamma-H2AX, and the phosphorylation site is a conserved serine four residues from the C-terminus, 139 in mammals and 129 in budding yeast. An antibody to gamma-H2AX reveals that the molecules form a gamma-focus at the DSB site. The gamma-focus increases in size rapidly for 10-30 min after formation, and remains until the break is repaired. Studies have revealed that small numbers of gamma-foci are present in cells even without the purposeful introduction of DNA DSBs. These cryptogenic foci increase in number during senescence in culture and aging in mice. This chapter presents techniques for revealing gamma-H2AX foci in cultured cells, in metaphase spreads from cultured cells, in tissues, and in yeast.

Animals↗

PubMed2004

DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.

The postreplicative repair of double-strand breaks (DSBs) is thought to require sister chromatid cohesion, provided by the cohesin complex along the chromosome arms. A further specialized role for cohesin in DSB repair is suggested by its de novo recruitment to regions of DNA damage in mammals. Here, we show in budding yeast that a single DSB induces the formation of a approximately 100 kb cohesin domain around the lesion. Our analyses suggest that the primary DNA damage checkpoint kinases Mec1p and Tel1p phosphorylate histone H2AX to generate a large domain, which is permissive for cohesin binding. Cohesin binding to the phospho-H2AX domain is enabled by Mre11p, a component of a critical repair complex, and Scc2p, a component of the cohesin loading machinery that is necessary for sister chromatid cohesion. We also provide evidence that the DSB-induced cohesin domain functions in postreplicative repair.

Cell Cycle Proteins↗

PubMed2004

Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break.

BACKGROUND: In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS: We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS: Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.

Chromatin↗