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

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. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. 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. Likewise, isolated nulcear 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 <I 5% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (~70%) were also sequestered within interior regions of the matrix. In contrast, 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 was measured in isolated nuclei before matrix fractionation. Furthermore, 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%). Our results support the hypothesis 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. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis 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.A number of investigators have shown that eucaryotic chromosomal DNA is organized into repeating supercoiled domains or loops (7,22,30,35,46,50). Recent results suggest that the DNA loops are constrained by attachment to components of the interphase nuclear matrix (4, 11,12,20,21,24,51,59) or its mitotic counterpart, the chromosome scaffold (5,25,48,51). While the precise relationships of isolated nuclear matrices to in situ nuclear structure have yet to be elucidated (9), a number of studies have demonstrated, depending on the conditions of isolation, a considerable degree of structural correspondence to in situ structure (15,19,31,40,53,62). In a particularly striking example, LaFond et al. (42) performed c...

The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

“…There is increasing evidence that eukaryotic origins are attached to the nuclear matrix (13,49) and that DNA replication takes place at the nuclear matrix (12,29,46,55). Several ARS elements, including ARSI and the H4 ARS, bind to the yeast nuclear scaffold (1).…”

DNA Replication Progresses on the Periphery of Nuclear Aggregates Formed by the BCL6 Transcription Factor

“…Moreover, the clustering and nuclear positioning of the DNA sequences replicated at individual replication foci are precisely maintained through the cell cycle and into subsequent generations, so that the BrdU foci persist as discrete structures even when replication does not occur (19,30,40,48,51). How this highly precise and reliable nuclear regionalization is achieved is still poorly understood, although the nuclear matrix is generally believed to be implicated (12,26,40,41,50,51).Both endogenous and overexpressed BCL6 localizes into discrete nuclear subdomains (3,11,17,29), hereafter referred to as nuclear aggregates. Using UTA-L cells, which express an epitope-tagged version of BCL6 upon removal of tetracycline from the culture medium, we previously reported that the large BCL6 aggregates formed upon BCL6 overexpression coincide * Corresponding author.…”

“…Moreover, the clustering and nuclear positioning of the DNA sequences replicated at individual replication foci are precisely maintained through the cell cycle and into subsequent generations, so that the BrdU foci persist as discrete structures even when replication does not occur (19,30,40,48,51). How this highly precise and reliable nuclear regionalization is achieved is still poorly understood, although the nuclear matrix is generally believed to be implicated (12,26,40,41,50,51).…”

Lipid phosphorylation in isolated rat liver nuclei Synthesis of polyphosphoinositides at subnuclear level

“…The nuclear lamina was purified with the same procedure employed for the nuclear matrix except that all buffers contained 5 mM dithiothreitol (DTT) and the isolated nuclei were digested with DNase I (14 U/mg DNA) and RNase A (80 pg/mg DNA) for 30 min in ice [14]. Aliquots of pm-detergent lamina were saved and the nuclear lamina was obtained with one final extraction in LS containing 0.4% Triton X-100.…”