Initiation and Continuation of DNA Replication Are Not Associated with the Nuclear Envelope in Mammalian Cells

Abstract

For determination of whether DNA replication is initiated at the nuclear envelope, synchronizedChinese hamster ovary cells labeled with [3HIthymidine were examined by electron microscope radioautography. The cells were synchronized initially by mitotic shake-off and held at the G1-S border by 5-fluorodeoxyuridine plus amethopterin. Cells were fixed at 1, 5, 10, and 30 min after the inhibitors were counteracted with [3H]thymidine.Radioautographic silver grains in each case were present over the more central parts of nuclei and were generally absent from the region of the nuclear envelope. We conclude that neither initiation nor continuation of DNA replication is associated with the nuclear envelope.An important facet of the problem of DNA replication in eukaryotes is whether initiation of replication occurs at points of attachment of the chromosome to the nuclear envelope. The question arises in part because of the evidence that the replicons of prokaryotic chromosomes initiate and then continue replication at points of attachment to the plasma membrane (see, for example, refs. 1-3). Several kinds of experiments on prokaryotes suggest that the enzymes and other factors necessary for DNA replication may form a complex that remains associated with the plasma membrane during chromosome replication (see ref. 4 for review).In studies of human amnion cells, Comings and Kakefuda (5) concluded from electron microscope radioautography that DNA replication is initiated at the nuclear envelope at the beginning of the period of DNA synthesis (S period). Contrary to this conclusion, Williams and Ockey (6) have obtained evidence by electron microscope radioautography that DNA replication at the beginning of the S period is not initiated at the nuclear envelope in Chinese hamster cells. According to them and to Huberman et al. (7), DNA synthesis late in the S period is highly concentrated near the nuclear envelope; this result is to be expected because replication of DNA in heterochromatin dominates the later part of the S period, and heterochromatin tends to be tightly condensed against the nuclear envelope. The results of Williams and Ockey (6) are to some degree supported by work of Erlandson and de Harven (8) for HeLa cells and of Blondel (9) for KB cells. In another eukaryote, Amoeba proteus, DNA replication is probably not associated with the nuclear envelope at any time during the S period (10).To answer the question of association of initiation or continuation of replication with the nuclear envelope in mammalian cells requires a very high degree of synchrony of the cultured cells. Many of the agents used for synchronization are incompletely effective. Ockey (11), for example, showed that amethopterin does not block cells from entering the S period. A high thymidine concentration (thymidine block) also fails to block cells at the G1-S border (12).We describe experiments here that minimize the shortcomings of current methods of synchronization. Our results strongly indicate that DNA replication is not initiated...

Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

“…The role of the nuclear membrane in the replication of cellular DNA is controversial. Several studies have found an association of nuclear membrane fractions with newly synthesized DNA by biochemical methods (9,12,14), whereas others have failed to find evidence for an association by cytological procedures (1,11,26). In the present study, two nuclear membrane fractions were isolated from adenovirus 2-infected cells by established procedures and were shown to contain newly synthesized DNA.…”

Localization of adenovirus DNA replication in KB cells

“…In 1969 Tremblay et al introduced a technique for the isolation of DNA-cell membrane complexes from bacteria based upon the cosedimentation of membrane-bound nascent DNA and Mg*+-Sarkosyl crystals (M-band). By analogy with bacterial systems, most cell fractionation experiments on the intracellular localization of DNA synthesis with the "M-band"-technique have been interpreted in favor of the attachment of replication sites to the nuclear membrane (Friedman and Mueller, 1969;Mizuno et al, 1971;Hanaoka and Yamada, 1971;Yamada and Hanaoka, 1973;autoradiographic investigations reveal that even after very short pulses with [3H]thymidine, DNA replication sites are randomly distributed throughout the nucleus (Williams and Ockey, 1970;Fakan et al, 1972;Huberman et al, 1973;Comings and Okada, 1973;Wise and Prescott, 1973).…”

Chromatin and DNA synthesis associated with nuclear membrane in germinating cotton.

“…Friedman and Mueller (8) suggested that replicating DNA in mammalian cells is attached to some nuclear site; this idea was supported by workers, using electron microscopic autoradiography, who concluded that DNA replication in mammalian cells is at least initiated at the nuclear envelope (9)(10)(11). Use of the same technique, however, has also shown that DNA replication is not initiated at the nuclear membrane in Chinese hamster cells (12,13) or mouse cells (14) and that any synthesis which appears to be membrane-bound occurs late in the S period and is believed to represent heterochromatin replication (12,15). Some anomalous results have been noted when biochemical methods were employed to clarify the problem of DNA replication in eukaryotes.…”

“…Recent work using radioautography tends to refute the concept of membrane-associated replication (12,13), while Hildebrand and Tobey (35) have published convincing biochemical data in support of membrane-associated replication. Since electron microscopy indicates large amounts of chromatin attached to nuclear membranes and pulse-chase studies indicate that the radioactivity remains membrane-associated, it may be that the membranes provide points of attachment which maintain chromatin in a proper configuration or spatial relationship.…”