In budding yeast replication origins, the 11-bp ARS consensus sequence is essential for interaction with the ORC. However, replication origins in other eukaryotic species, including fission yeast, do not appear to contain a short essential sequence (15
) and it has not been known whether the ORC is located at chromosomal replication origins. The present study demonstrated that a fission yeast ORC subunit and an Mcm protein are specifically localized at chromosomal replication origins. Orp1p is located at the ars2004
loci throughout the cell cycle, while SpMcm6p is associated with these origins only in the G1
and S phases. To our knowledge, this is the first indication of preferential localization of the ORC and Mcm proteins at the chromosomal replication origins in eukaryotic species except for budding yeast.
The CHIP assay finding that Orp1p was localized at ars2004
but not at non-ARS regions (Fig. ) suggests that a certain sequence or DNA structure in the replication origins is recognized by the fission yeast ORC. Although there is no extensive sequence homology between these two ARS fragments, the regions required for the ARS activities are rich in adenines on a DNA strand (19
). Orp1p was preferentially located within a 1-kb region of ars2004M
(Fig. ) in which the replication initiation site had been mapped (50
contains two adenine/thymine-rich regions, regions I and III, which are essential for ARS activity (51
). Thus, an adenine-thymine-rich stretch is the primary candidate for the ORC-binding site. Segments 2 and 3, which are about 1 kb apart and proximal to regions I and III, respectively, were similarly amplified in the CHIP assay. The ORC would interact either with the unique region at the center of segments 2 and 3 or with multiple regions within ars2004M
. Since the central region of ars2004M
is not essential for ARS activity (51
), it seems likely that ORCs interact with both regions I and III. Regions I and III can be functionally replaced with repeats of AAAT, but not AAT or AT, suggesting that various sequences with three or more consecutive adenines, rather than mere AT richness, are required for interaction with the ORC. These results suggest that the mechanism of interaction of the fission yeast ORC with replication origins might be different from that of the budding yeast ORC. Recently, fission yeast Orp4, a homologue of budding yeast Orc4, has been shown to contain AT-hook motifs that are involved in interaction with the minor groove of AT tracts in DNA (12
). The N-terminal domain of Orp4p, with nine AT-hook motifs, specifically binds to the fission yeast ars1
fragment that contains adenine-thymine stretches. The DNA binding activity of Orp4p might participate in recognition of fission yeast replication origins. Further genetic and biochemical studies are required for better understanding of the nature of the fission yeast ORC.
The results of MNase digestion (Fig. ) suggest that some Orp1p molecules are associated with insoluble nuclear components. We observed that chromosomal origin regions were not readily released from the insoluble fraction by sonication (48
), suggesting that Orp1p in the insoluble fraction interacts with both chromatin and insoluble components. Interaction of the ORC with insoluble nuclear components might be involved in the localization of origins at certain nuclear structures. It has been reported that DNA replication in mammalian cells occurs in a distinct nuclear structure, called a replication factory, which is tightly attached to the nuclear matrix (27
). On the other hand, the budding yeast ORC has been shown to be involved in cellular functions other than chromosome replication, such as silencing of mating type control (39
) and mitotic chromatin condensation (17
). The relationship of these functions to the subnuclear localization of the ORC remains to be elucidated.
In addition to Orp1p, SpMcm6p is preferentially located within an about 1-kb region of ars2004
, as shown by the CHIP assay. SpMcm6p was associated with the ars2004
loci only during the G1
and S phases, at the same time as when it was associated with chromatin (compare Fig. and ). In a similar period of the cell cycle, SpMcm6p was detected as distinct foci on spread nucleoids (Fig. ), suggesting that the foci contain replication origins associated with SpMcm6p. The number of SpMcm6 foci is, at most, 50, even in cells arrested early in S phase by addition of HU (48
). Thus, the number of SpMcm6 foci per nucleoid is about 10 times smaller than the number of putative replication origins (50). Multiple replication origins that are associated with the ORC and Mcm proteins could be clustered in the foci. The clustering might facilitate effective activation and inactivation of a set of replication origins by cell cycle signals. Budding yeast Mcm7p has also been shown to be found as distinct foci on spread chromatin by immunostaining (60
). In higher eukaryotic cells, however, the entire chromosome regions are immunostained with anti-Mcm antibodies (33
). It remains to be solved whether higher eukaryotic Mcm proteins are located at specific regions of the chromosome or exist along the entire chromosome. Even in the former case, several thousand Mcm signals might not be separable as distinct foci due to possible overlapping.
For association of SpMcm6p with chromatin, Cdc18p, a homologue of Cdc6p, was required (Fig. ). This is consistent with the requirement of Cdc6p, as well as the ORC, for chromatin loading of Mcm proteins in budding yeast and Xenopus
egg extract (14
). The ORC, Cdc6p, and Mcm proteins become sequentially associated with replication origins to form Pre-RCs (18
). A certain temperature-sensitive mutation in orp1
impaired the association of Mcm6p with chromatin at the restrictive temperature (4a
), suggesting dependency of chromatin-association of Mcm proteins on the ORC function. After loading of Mcm, however, the budding yeast and Xenopus
ORCs and Cdc6p can be removed from chromatin without removing Mcm proteins, suggesting that Mcm proteins, once loaded, are associated with some other component (18
). The CHIP assay with anti-SpMcm6p showed that segment 2 was more efficiently amplified than segment 3 in ars2004M
, while both segments were similarly amplified by immunoprecipitation with anti-FLAG antibody. The location of SpMcm6p within ars2004
might not be the same as that of the ORC. In human cells, the ORC and Mcm proteins are not colocalized within a 500-bp region of the chromosome (53
Mcm proteins, components of Pre-RCs in budding yeast, are relocated at nonorigin regions with kinetics similar to those of DNA polymerase ε, which moves with replication forks (4
). Although we have not observed significant association of SpMcm6p with non-ARS fragments (Fig. ), transient localization of Mcm proteins in non-ARS regions might not be detected due to faster fork movement under our conditions. On the other hand, Mcm proteins might be involved in possible unwinding of replication origins, which results in association of replication protein A (RPA), a single-stranded DNA binding protein complex (61
). Ishimi has shown that a hexamer complex composed of human Mcm4, -6, and -7 proteins exhibits weak DNA helicase activity (30
). Possible Mcm protein DNA helicase activity could be responsible for origin unwinding, replication fork movement, or both. Since association and dissociation of Mcm proteins from replication origins could be key reactions in the regulation of replication, more-detailed analysis of molecular mechanisms of interaction of Mcm proteins with replication origins is necessary. Fission yeast replication origins composed of functional elements located far from each other might be suitable for analysis of the assembly of various protein factors during the process of initiation of replication.