Abstract
Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.
Highlights
In a eukaryotic cell committed to duplication, chromosomal DNA replication initiates at many sites called origins of DNA replication
We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction
DmORC binds to DNA fragments of variable sequence composition Our initial goal was to determine a high-affinity DmORCbinding site in ACE-3 and ori-b using DNase 1 protection
Summary
In a eukaryotic cell committed to duplication, chromosomal DNA replication initiates at many sites called origins of DNA replication. One of the most tractable systems for studying origin choice and ORC localization in metazoans is provided by the DNA amplicons in the follicle cells surrounding the developing oocyte in Drosophila melanogaster (Calvi and Spradling, 1999) In these somatic cells, the chorion genes on the third and X chromosome undergo site-specific DNA amplification to allow for a rapid increase in the number of templates for later transcription of the egg shell genes. A similar requirement for origin function in bacteriophage l suggests that (À) supercoiled DNA may be broadly important for origin function (Schnos et al, 1988) We tested this parameter for the DmORC–DNA complex and found that (À) supercoiling dramatically increases DmORC affinity but not specificity to DNA. The implication of these studies to the general usefulness of the replicon hypothesis in its simple form is discussed
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