Organization of early and late replicating DNA in human chromosome territories

It has been suggested that DNA organized into replication foci during S-pha se remains stably aggregated in non-S-phase cells and that these stable agg regates provide fundamental units of nuclear or chromosome architecture [C, Meng and R. Berezney (1991) J, Cell Biol. 115, 95a; E, Sparvoli et at (199 4) J. Cell Sci, 107, 3097-3103; D, A. Jackson and A. Pombo (1998) J. Cell B iol, 140, 1285-1295; D, Zink et al, (1998) Hum, Genet, 112, 241-251], To te st this hypothesis, early and late replicating DNA of human diploid fibrobl asts was labeled specifically by incorporating two different thymidine anal ogs [J. Aten (1992) Histochem, J, 24, 251-259; A, E, Visser (1998) Exp, Cel l Res. 243, 398-407], during distinct time segments of S-phase, On mitotic chromosomes the amount and spatial distribution of early and late replicati ng DNA corresponded to R/G-banding patterns, After labeling cells were grow n for several cell cycles. During this growth period individual replication labeled chromosomes were distributed into an environment of unlabeled chro mosomes, The nuclear territories of chromosomes 13 and 15 were identified b y additional chromosome painting, The distribution of early and late replic ating DNA was analyzed for both chromosomes in quiescent (G(0)) cells or at G(1), Early and late replicating DNA occupied distinct foci within chromos ome territories, displaying a median overlap of only 5-10%. There was no di fference in this regard between G(1) and G(0) cells, Chromosome 13 and 15 t erritories displayed a similar structural rearrangement in G(1) cells compa red to G(0) cells resulting in the compaction of the territories. The findi ngs demonstrate that early and late replicating foci are maintained during subsequent cell cycles as distinctly separated units of chromosome organiza tion, These findings are compatible with the hypothesis that DNA organized into replicon clusters remains stably aggregated in non-S-phase cells, (C) 1999 Academic Press.