Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.

Open Access
Publisher Website
Google Scholar
Abstract
Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. The distribution of these DNA components with the peripheral nuclear lamina vs. more internally localized structural elements of isolated nuclear matrix was determined. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained < 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase .alpha. (.apprx. 70%) were also sequestered within interior regions of the matrix. A majority of the endogenous DNA template sites for DNA polymerase .beta. (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities were measured in isolated nuclei before matrix fractionation. Isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase .alpha. endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding .beta. polymerase activities (47-52%). The hypothesis is supported that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. The sites of nuclear DNA polymerase .beta.-driven DNA synthesis apparently are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.