PREFERENTIAL CROSS-LINKING OF MATRIX-ATTACHMENT REGION (MAR) CONTAINING DNA FRAGMENTS TO THE ISOLATED NUCLEAR MATRIX BY IONIZING-RADIATION

The sequences that anchor DNA, matrix-attachment regions (MARs), can b e 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 devel oped an in vitro binding assay by using nuclear matrices isolated from murine erythroleukemia cells by high-salt extraction of DNase I-diges ted nuclei. The cross-linking of nuclear matrix protein to DNA fragmen ts containing kappa-immunoglobulin (kappa-Ig) or an hsp70 MAR was stud ied. Fragments of pBR322 of similar size to the MAR-containing fragmen ts served as non-MAR controls. Two types of experiments were conducted : type A in which nuclei were irradiated and nuclear matrices were iso lated and assayed for the binding of exogenous P-32-labeled DNA fragme nts, and type B in which mixtures of isolated nuclear matrices and [P- 32]DNAs were irradiated and assayed for binding. Poly(dAT) served as a competitor in the binding assays, because it eliminated nonspecific b inding of DNA to the nuclear matrix and revealed the radiation-induced increase in tightly bound DNA. When nuclear matrices were isolated fr om irradiated nuclei (0-200 Gy) and incubated with the kappa-Ig MAR fr agment in the absence of poly(dAT) (type A experiments), much nonspeci fic, non-dose-dependent binding was observed. With poly(dAT) in the in cubation mixture, a dose-dependent decrease (p < 0.001) in the binding was revealed, indicating a radiation-induced loss of available bindin g sites, perhaps due to the cross-linking of endogenous sequences. The pBR322 fragment did not show a similar loss of binding sites. Irradia tion of mixtures of isolated nuclear matrices and end-labeled fragment s (type B experiments) allowed the study of radiation-induced cross-li nking of exogenous fragments to the matrices. If poly(dAT) was present during irradiation, nonspecific binding was eliminated; however, no s ignificant increase (p = 0.5) in the specific binding of the DNA to th e nuclear matrix was observed. In contrast, if poly(dAT) was added aft er 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 th e pBR322 fragments. The results indicate that the specific interaction of MARs with proteins of the nuclear matrix provides a radiation-sens itive substrate for the formation of DNA-protein cross-links.