CHROMATIN-ASSEMBLY-FACTOR-1 (CAF-1) COLOCALIZES WITH REPLICATION FOCIIN HELA-CELL NUCLEI

In the S-phase of the eukaryotic cell cycle, newly replicated DNA is a ssembled into chromatin. I used indirect immunofluorescence microscopy to localize the sites of chromatin assembly in respect to DNA replica tion. Replication foci in the nuclei of permeabilized HeLa cells were labeled by incorporation of biotin-16-dUTP and detected by fluorescent streptavidin. Prelabeling of replication foci in vivo with bromodeoxy uridine showed that replication in permeabilized cells proceeds at pre existing replication forks. The localization of chromatin assembly fac tor 1 (CAF-1) was determined with subunit-specific monoclonal antibodi es. CAF-1 is not detectable in mitotic cells and is detectable only at background levels in about 60% of all interphase nuclei. The other in terphase nuclei show an intense punctate immunostaining of CAF-1. Thes e sites of CAF-1 colocalize with replication foci during all stages of the S-phase. No other discrete sites of CAF-1 are observed. Human rep lication protein A (RPA) colocalizes with these replication/chromatin assembly sites. In addition, extra nuclear sites of RPA are observed t hat probably represent prereplication foci, poised for initiation of D NA replication. (C) 1995 Academic Press, Inc.