Early-replicating heterochromatin

Abstract

Euchromatin, which has an open structure and is frequently transcribed, tends to replicate in early S phase. Heterochromatin, which is more condensed and rarely transcribed, usually replicates in late S phase. Here, we report significant deviation from this correlation in the fission yeast, Schizosaccharomyces pombe. We found that heterochromatic centromeres and silent mating-type cassettes replicate in early S phase. Only heterochromatic telomeres replicate in late S phase. Research in other laboratories has shown that occasionally other organisms also replicate some of their heterochromatin in early S phase. Thus, late replication is not an obligatory feature of heterochromatin.

Figure 1

The central portions of the centromeres are replicated in early S phase. (A) The cdc10 temperature block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 4 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. (B) The hydroxyurea (HU) block and release procedure (Kim and Huberman 2001) was used to synchronize passage through S phase. This experiment is identical to the one shown in Figure 7 in Kim and Huberman (2001). The Southern membrane used in that experiment was stripped and rehybridized with probes specific for the cnt sequences. Note that the cells in the zero-minute time point had been exposed to HU for 5 h at 25°C.

Figure 2

Structures of the major fission yeast mating-type locus configurations. These diagrams are based on nucleotide sequence information (GenBank accession nos. AL035065, AL356712, AL353012, and U57841) and on previous studies describing the derivation of h^−S and h^+N by recombination from h⁹⁰ (Beach and Klar 1984). All three configurations are shown at the same scale. Restriction fragments studied for replication timing are indicated by horizontal boxes in the upper portions of the diagrams. The position of the hybridization probe used to identify each restriction fragment is shown as a thick black line immediately below the restriction fragment. The horizontally striped arrow in the CEN homology region represents dg sequences. The vertically striped arrow represents dh sequences, and the black arrowhead indicates an otr repeat that is frequently located between dh and dg sequences. See the text for additional description.

Figure 3

The mating-type region is replicated in early S phase. Locations of the studied restriction fragments are shown in Figure 2. (A) The cdc10 block and release procedure was used to synchronize the h^−S strain. This experiment is identical to the one shown in Kim and Huberman (2001). The membrane was stripped and rehybridized with probes for the indicated restriction fragments. (B) The same procedure was used to synchronize the h^+N strain. The experiment is the same as in Figure 5B in Kim and Huberman (2001). The membrane was stripped and rehybridized with probes for the indicated restriction fragments.

Figure 4

Early replication of the mating-type region. (A) The HU synchronization procedure was used with the h^−S strain. This experiment is identical to the one shown in Figure 7 in Kim and Huberman (2001). The membrane was stripped and rehybridized with a probe for restriction fragment 2 (Fig. 2). (B) The centrifugal elutriation procedure was used with the h^+N strain. This experiment is the same as in Figures 1 and 2 in Kim and Huberman (2001). The membrane was stripped and rehybridized with probes for the indicated regions. See the text and Materials and Methods for further details on the probes used.