The sites of nascent DNA synthesis were compared
with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells
were compared with the distribution of PCNA by
indirect immunofluorescence microscopy and laser
scanning confocal microscopy (LSCM). In common
with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe
coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly,
nuclei which had been isolated from quiescent
cultures of HDF were innoculated into cell-free
extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated
at multiple discrete spots some 15min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.Brunel Open Access Publishing Fun
