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.
Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, Building 37, Room 6124, Bethesda, MD 20892, USA.
Robert
R
Shroff
Ayelet
A
Arbel-Eden
Duane
D
Pilch
Grzegorz
G
Ira
William M
WM
Bonner
John H
JH
Petrini
James E
JE
Haber
Michael
M
Lichten
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