HIGH RESOLUTION ANALYSIS OF REPLICATION FOCI BY CONVENTIONAL FLUORESCENCE MICROSCOPY.
I. A STUDY OF COMPLEXITY AND DNA CONTENT OF THE FOCI
V. O. Chagin, 1 Yu. M. Rozanov, L. V. Solovjeva, N. V. Tomilin
Institute of Cytology RAS, St. Petersburg, Russia;
1 e-mail: chaga@link.cytspb.rssi.ru
Newly replicated DNA segments (RDS) have been shown to form discrete foci in the mammalian nucleus. Comparison
of the number of such foci in formaldehyde-fixed cell nucleus with estimated number of simultaneously active replication forks (RF) suggests
that each replication focus contains a cluster of about 10 to 20 closely associated RF. That implied the cluster of synchronously activated
replicons as the primary unit of mammalian DNA replication. It still remains unclear whether such clustering of RF does mean adjacency
of the replicons in a genomic location (structural clustering, model 1), or it arises from transient clustering of the replicons from different
DNA domains at the functioning replicaton machinery (functional clustering, model 2). In this study we used conventional fluorescence
microscopy of the hypotonically treated nuclei preparations to investigate replication foci at the optical resolution limit. Human K562 cells
were labeled with 5'-iododeoxyuridine for different time periods. We synchronized the cell culture with hydroxyurea to be able to measure
an average increase in DNA content during labeling period using DNA cytometry. Under these conditions, RDS appear as multiple small
foci (mini-foci, MF). Further studies revealed that most of such mini-foci of replication represent optical diffraction spots, which are standard
in size and different in brightness. The number of the "spots" and variation of their brightness mostly depend on the extent of hypotonic
treatment. Flow cytometry control of the synchronized cells peak movement allowed us to measure mean DNA content of the MF. In case
of most effective hypotonic treatment, a MF contains about 40 Kbp of labeled DNA, and the general number of the MF approaches the
number of replicons that are simultaneously active in a given moment of S-phase. Influence of the effect 6f hypotonic treatment on overall
number of observed MF suggests that replication foci in early and mid S-phase cells do not represent stable structures, but rther arise from functional clustering of comparatively distant replicating regions, thus supporting model 2.
Back
Contents
Main
|
