Our long-range objectives are to understand how replication of mammalian chromosomes is controlled. Utilizing a wide variety of replicon mapping strategies, we and others have characterized the origin of replication in the Chinese hamster DHFR locus. The origin is situated in the 55 kb spacer region between the DHFR and 2BE2121 genes, which are convergently transcribed in the early S period when the DHFR origin is activated. Although replication can initiate at any one of several potential sites scattered throughout the intergenic spacer, two subregions or sites (ori-beta and ori-gamma) are preferred. Depending upon whether the initiation reaction is examined by a PCR-based nascent strand abundance assay or by 2-D gel replicon mapping methods, ori-beta and ori-gamma are suggested to be either highly or only moderately preferred. However, each of these sites displays a pronounced nuclease hypersensitive site in the pre-replicative state, which is dissipated as cells enter the S period. We have shown that removal of ori-beta by homologous recombination has no overt effect on origin activity in the intergenic spacer, suggesting that if ori-beta contains a replicator, it must be redundant. In contrast, deletion of either the 5' or 3' ends of the DHFR gene eliminates origin activity in this locus. Our working hypothesis is: 1) that there is a hierarchy of potential (and therefore redundant) replicators in the intergenic region, with ori-beta and ori- gamma being preferred; 2) that initiation at ori-beta and ori-gamma (or at other sites) is effected by interaction with an initiator complex; and 3) that activation of the origin in the early S period depends upon transcription of the DHFR and/or 2BE2121 genes during the G1/S transition.
Specific aims are: 1) To determine why the nascent strand abundance assay suggests that ori-beta and ori-gamma are major start sites, while 2-D gel approaches suggest that ori-beta and ori-gamma are only somewhat preferred over other sites in the spacer. The same preparations of replication intermediates will be analyzed by the PCR- based assay and by a quantitative neutral/alkaline 2-D gel approach to determine the source of the differing views of initiation in this locus. 2) To define the minimal sequences required to restore origin activity to a cell line that lacks the intergenic spacer. 3) To define the elements in the promoter of the DHFR gene that are required for origin activity, using a homologous recombination approach to delete or replace selected sequences. 4) To determine whether the 3' end of the DHFR genes prevents interference with initiation, or contains sequence elements actively required for initiation, using homologous recombination to modify the 3' end. 5) To determine the distribution of ORC and MCM proteins in the DHFR domain vis-a-vis initiation sites, using a chromatin immunoprecipitation (CHIP) assay.
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