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.
Shroff
Robert
R
Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, Building 37, Room 6124, Bethesda, MD 20892, USA.
Arbel-Eden
Ayelet
A
Pilch
Duane
D
Ira
Grzegorz
G
Bonner
William M
WM
Petrini
John H
JH
Haber
James E
JE
Lichten
Michael
M
D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D053938:DNA Fragmentation / Q000502:physiology*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D047468:Immunoprecipitation; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D020033:Protein Isoforms; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific