Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Abstract

In mouse cells (line P815), newly synthesized DNA labeled for [20][21][22][23][24][25][26][27][28][29][30] sec during exponential growth is found by electron microscope autoradiography at sites throughout the cell nucleus. These sites are relatively more concentrated in the peripheral region of the nucleus (averaged over a random population of S-phase cells), probably reflecting a higher local concentration of DNA in this region. Newly synthesized DNA is not preferentially associated with purified nuclear envelopes, but is found in a fraction of the chromosomal deoxynucleoprotein whose buoyant density in CsCl after formaldehyde treatment is about 1% lower than that of the deoxynucleoprotein peak.Kinetics experiments suggest that this material is a precursor of mature deoxynucleoprotein; it may represent regions of deoxynucleoprotein containing replicating DNA and the additional proteins involved in DNA replication. Other complexes of newly replicated DNA that are found in the interphase after phenol extraction of nuclei are formed during the extraction procedure, probably due to the partially single-stranded nature of replicating DNA, and do not appear to exist in vivo.The many studies on the location of replication sites of chromosomal DNA in the eukaryotic nucleus have led to divergent conclusions. Electron microscope autoradiography (1-3) shows that replication sites of chromosomal DNA are distributed throughout eukaryotic nuclei, and do not appear to be associated with any specific morphological structure. The peripheral replication sites in cells released from synchronizing inhibitors (4) may not represent normal S-phase initiation (5).In contrast, most (6,7,8) [but not all (9)] biochemical studies show that newly replicated DNA is preferentially associated with the nuclear envelope. Newly replicated DNA is often (6, 10-12), but not always (13,14), localized in the interphase after phenol or chloroform-isoamyl alcohol extraction. It sometimes associates preferentially with crystals of detergent in sucrose gradients (7,30). The interpretation of these observations has been influenced by models of membrane-associated replication of bacterial (15) and phage (16) DNA; the phage DNA-membrane association may, however, be related to transcription rather than replication (17).We present here studies designed to resolve these discrepancies and to identify by two independent methods, electron microscope autoradiography and cell fractionation, the sites of chromosomal DNA synthesis in exponentially growing mouse cells. Eukaryotic chromosomal DNA replicates at 0.5 (18) to 1-2 (19) Mum/min; within the nucleus, therefore, a labeled precursor newly incorporated into DNA could be displaced as much as 1 Mm away from its site of incorporation after 1 min. We have used labeling periods of 20-30 sec, which are about the lower practical limit for present techniques of electron microscope autoradiography; labeled molecules incorporated at the beginning of this period are unlikely to be displaced more than 0.5 ,m from the...

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

“…Our results strongly indicate that DNA replication is not initiated at the nuclear envelope at the start of the S period and that, in agreement with results of Fakan et al (13) Chinese hamster ovary (CHO) cells were cultured in monolayers as described (12) and synchronized by the mitotic shake-off method (14). The shake-off populations consisted of 96-98% mitotic cells.…”

“…Finally, we released cells from the inhibitors with 1-and (13) and Huberman et al (7), our experiments show that the enzymatic machinery for DNA replication is not attached to the nuclear envelope, i.e., the replication forks are not attached to membrane. Thus, the mechanism of DNA replication in higher eukaryotes may be significantly different from the mechanism in prokaryotes.…”

Localization of adenovirus DNA replication in KB cells

“…Since replicating Ad&DNA molecules contain long stretches of single-stranded DNA, these regions may associate preferentially with the nuclear membrane or with the M-band complex, as has been suggested (Fakan et al, 1972;Huberman et aE., 1973). Control experiments, however, indicate that the association of pulse-labeled Ad5-DNA with the M-band complex is probably not due to the single-stranded regions in replicating Ad5-DNA molecules.…”

“…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).…”

Accumulation of an Intermediate in DNA Synthesis by HEp.2 Cells Treated with Methyl Methanesulfonate

“…Cells were harvested and washed in phosphate-buffered saline (11), washed in 0.15 M NaCl + 0.015 M sodium citrate, and resuspended in saline-citrate at approximately 4.0 X 106/ml. Single-stranded, newly synthesized DNA is preferentially adsorbed to the interface of organic solvent extractions (13,14). Our preparations must, therefore, be done at high cell density (15), which makes it possible to avoid the losses in recovery reported by Hanawalt and Ray (16).…”