Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences.
Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
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Galande
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Kohwi-Shigematsu
D034961:Antigens, Nuclear; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D000072200:Ku Autoantigen; D009687:Nuclear Proteins / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D009687:Nuclear Proteins; D011065:Poly(ADP-ribose) Polymerases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen