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Abstract. We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of >90 % of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus. DSPITE considerable progress in defining specific molecular components involved in eucaryotic DNA replication such as DNA polymerases a, di, and DNA primase (Kornberg, 1988), our understanding of native replicational sites and their structural organization and associations in the cell nucleus has lagged behind. Most previous studies designed to localize the sites of DNA replication in eucaryotic cells have used autoradiographic microscopy. These techniques have been very useful in determining cells active in DNA replication and the general distribution of replication sites in the cell nucleus over peripheral versus internal sites and over condensed heterochromatin versus diffuse euchromatin (Hay and Revel, 1963;Milner, 1969; Huberman et al., 1973;Fakan and Hancock, 1974;Fakan, 1978;Smith et al., 1984). The level of resolution of these techniques, however, severely limits their potential usefulness for studying the structural organization of individual replicational sites in the cell nucleus.This has prompted us to explore more sensitive and higher-resolution approaches to this problem. Langer et al. (1981), who first synthesized biotin-labeled nucleotides for use as nucleic acid-affinity probes also demonstrated that 5-([N-biotinamidocaproyl]-3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (biotin-ll-dUTP) ~ is effectively incorporated Dr. Nakayasu's present address is Department of Medical Biochemistry, Shiga, University of Medical Sciences, Seta, Otsu, 52021, Japan. Address correspondence to Dr. Ronald Berezney, Department of Biological Sciences, State University of New York, Buffalo, NY 14260. Abbreviations used in this paper:biotin-lI-dUTP, 5-([N-biotinamidocaproyl]-3-aminoallyl)-2'-deoxyuridine-5'-triphosphate; BrdU, 5-bromodeoxyuridine; glycerol buffer, 20 mM Tris-HCI, pH 7.4, 25% glycerol, 5 mM MgCt2, 0.5 mM EGTA, 0.5 mM PMSF; TBS buffer, 10 mM Tris-HC1, into DNA by a variety of DNA polymerases, including the...

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10-100 adjacent replicons (each approximately 100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought - so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45-60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase.

Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of ∼1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take ∼45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or ∼6 average-sized replicons. Double pulse–double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.