Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins.
BACKGROUND
Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC.


RESULTS
The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC-IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC-IPs correspond closely to those necessary for in vivo ARS activity.


CONCLUSIONS
Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC-binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.
Takahashi
T
T
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043 Japan.
Masukata
H
H
D000225:Adenine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D011233:Precipitin Tests; D021122:Protein Subunits; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D013941:Thymine / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D000225:Adenine; D013941:Thymine