Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR.
Apoptotic DNA degradation could be initiated by the accumulation of single-strand (ss) breaks in vulnerable chromatin regions, such as base unpairing regions (BURs), which might be preferentially targeted for degradation by both proteases and nucleases. We tested this hypothesis in anti-Fas-treated apoptotic Jurkat cells. Several nuclear proteins known for their association with both MARs and the nuclear matrix, that is, PARP, NuMA, lamin B and SATB1, were degraded, but the morphological rearrangement of the BUR-binding SATB1 protein was one of the earliest detected changes. Subsequently, we have identified several genes containing sequences homologous to the 25 bp BUR element of the IgH gene, a known SATB1-binding site, and examined the integrity of genomic DNA in their vicinity. Multiple ss breaks were found in close proximity to these sites relative to adjacent regions of DNA. Consistent with our prediction, the results indicated that the initiation of DNA cleavage in anti-Fas-treated Jurkat cells occurred within the BUR sites, which likely became accessible to endonucleases due to the degradation of BUR-binding proteins.
Liu
Q Y
QY
Apoptosis Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Ribecco-Lutkiewicz
M
M
Carson
C
C
Testolin
L
L
Bergeron
D
D
Kohwi-Shigematsu
T
T
Walker
P R
PR
Sikorska
M
M
D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D004277:DNA, Single-Stranded; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D007700:Kinetics; D036961:Matrix Attachment Region Binding Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
D002843:Chromatin; D004277:DNA, Single-Stranded; D036961:Matrix Attachment Region Binding Proteins; D009687:Nuclear Proteins; C076189:SATB1 protein, human