Balasubramaniam U, Oleinick N L
Department of Environmental Health Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942, USA.
Biochemistry. 1995 Oct 3;34(39):12790-802. doi: 10.1021/bi00039a040.
The sequences that anchor DNA, matrix-attachment regions (MARs), can be identified by their specific and preferential binding to the nuclear matrix. This microenvironment may be hypersensitive to the formation of ionizing radiation-induced DNA damage, including DNA-protein cross-links (DPC). To examine the induction of DPC at or near MARs, we developed an in vitro binding assay by using nuclear matrices isolated from murine erythroleukemia cells by high-salt extraction of DNase I-digested nuclei. The cross-linking of nuclear matrix protein to DNA fragments containing kappa-immunoglobulin (kappa-Ig) or an hsp 70 MAR was studied. Fragments of pBR322 of similar size to the MAR-containing fragments served as non-MAR controls. Two types of experiments were conducted: type A in which nuclei were irradiated and nuclear matrices were isolated and assayed for the binding of exogenous 32P-labeled DNA fragments, and type B in which mixtures of isolated nuclear matrices and [32P]DNAs were irradiated and assayed for binding. Poly(dAT) served as a competitor in the binding assays, because it eliminated nonspecific binding of DNA to the nuclear matrix and revealed the radiation-induced increase in tightly bound DNA. When nuclear matrices were isolated from irradiated nuclei (0-200 Gy) and incubated with the kappa-Ig MAR fragment in the absence of poly(dAT) (type A experiments), much nonspecific, non-dose-dependent binding was observed. With poly(dAT) in the incubation mixture, a dose-dependent decrease (p < 0.001) in the binding was revealed, indicating a radiation-induced loss of available binding sites, perhaps due to the cross-linking of endogenous sequences. The pBR322 fragment did not show a similar loss of binding sites. Irradiation of mixtures of isolated nuclear matrices and end-labeled fragments (type B experiments) allowed the study of radiation-induced cross-linking of exogenous fragments to the matrices. If poly(dAT) was present during irradiation, nonspecific binding was eliminated; however, no significant increase (p = 0.5) in the specific binding of the DNA to the nuclear matrix was observed. In contrast, if poly(dAT) was added after irradiation, in addition to the elimination of nonspecific binding, a radiation dose-dependent increase in binding was revealed for both the kappa-Ig MAR and the hsp MAR (p < 0.001), but not for either of the pBR322 fragments. The results indicate that the specific interaction of MARs with proteins of the nuclear matrix provides a radiation-sensitive substrate for the formation of DNA-protein cross-links.
