Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin.
A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.
Savic
Velibor
V
Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Yin
Bu
B
Maas
Nancy L
NL
Bredemeyer
Andrea L
AL
Carpenter
Andrea C
AC
Helmink
Beth A
BA
Yang-Iott
Katherine S
KS
Sleckman
Barry P
BP
Bassing
Craig H
CH
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D016193:G1 Phase / Q000502:physiology; D019673:Genes, T-Cell Receptor alpha / Q000235:genetics; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D013950:Thymus Gland / Q000166:cytology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004247:DNA; D004268:DNA-Binding Proteins; D004720:Endonucleases; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C508937:MDC1 protein, mouse; C576325:Atm protein, mouse; C473337:Dclre1c protein, mouse; C475961:gamma-H2AX protein, mouse