Quantitative analysis of GFP-PCNA foci appearing adjacent to nascent DNA during S-phase
progression
In order to quantitatively evaluate what percentage of GFP-labelled foci appeared in the vicinity of
previously replicated nascent DNA during S-phase progression, we analysed 913 GFP-labelled foci in
randomly selected double-labelled nuclei 90 minutes after microinjection. As replication sites are active on
average for about 45-60 minutes (Ma et al., 1998; Manders et al., 1992; Manders et al., 1996), one would
expect that during this time period most foci actively incorporating Cy3-dUTP in the beginning, finished
replication, and that initiation at new sites took place. The data revealed that at 90 minutes after
microinjection 93.62.4% of GFP-labelled foci were located at distances of less than 1 m from Cy3labelled DNA.
Statistical analysis of the probability of observing a high degree of colocalization in doublelabelled daughter nuclei
For testing whether 85% of colocalization was observed in a fraction of nuclei due to random crowding of
foci we focused on the second half of S phase. On average, during the second half of S phase we observed
about 60 labelled DNA foci and about 60 GFP-PCNA foci in a given nuclear plane. Each class of foci
occupied about 10% of the relevant nuclear regions, which were during the second half of S phase; mainly
regions at the nuclear and nucleolar peripheries with widths of about 1 m. In order to calculate the
probability (P) for a high level of colocalization if DNA foci and GFP-PCNA randomly occupied the
nuclear and nucleolar peripheries, we subdivided these regions into fields corresponding to locations that
could be occupied by a DNA focus. In this manner, the nuclear and nucleolar peripheries of an ellipsoidal
model nucleus (length: 20 m, width: 10 m) containing two nucleoli with a diameter of 4 m each were
subdivided into 600 fields within a given nuclear plane. Our assumption was that if 60 DNA foci occupy
10% of the relevant nuclear regions in a given nuclear plane 600 fields are at least available. The
probability (P) that a DNA focus and a GFP-PCNA focus colocalize in a given field was determined by a
binomial distribution
 y  x - y
   

n  y - n
x
   p ( x, y, n) . x refers to the number of fields, y to the
 y
number of occupied fields (DNA foci already present in that region), and n to the number of fields occupied
by a DNA focus and a GFP-PCNA focus (colocalization). Using the numbers given above, on average
about 30% of DNA foci and GFP-PCNA foci would be expected to colocalize with each other. The
probability that 85% of DNA and GFP-PCNA foci colocalize would be 1050. This result has been obtained
assuming that at each site, only 80% of the pixels of the Cy3- and GFP-labelled foci colocalize. In reality,
often a higher degree of colocalization has been observed (see e.g. Fig. 8b). Given this higher degree of
colocalization, the expected probability would be even lower than 10 50.