In an effort to delineate the molecular events that control initiation of DNA synthesis in mammalian chromosomes, we have studied initiation and progression of DNA replication within the amplified dihydrofolate reductase (DHFR) domains of the methotrexate-resistant Chinese hamster cell strain, CHOC 400. Hybridization studies using nascent DNA synthesized during the onset of S phase in whole or permeabilized cells has shown that replication of the amplified DHFR domains is initiated within a 4.3 kb Xba I fragment that maps 14 kb downstream from the amplified DHFR gene. We propose to complete physical characterization of the initiation region by mapping and sequencing 11 kb of DNA surrounding the DHFR origin of replication. We will simultaneously conduct strand specific hybridization studies with Okazaki fragments in order to precisely locate the initiation sites within the DHFR origin fragment. In addition, we will intrate the sensitivity of template sequences to crosslinking agents and nucleases in permeabilized G1/S cells in order to define conditions for limiting replication in vitro to initiation events. These localization studies will be correlated with an electron microscopic examination of initiation events within the amplified domain. Preliminary EM observations have revealed unusual secondary structures that likely represent novel intermediates in initiation of DNA synthesis. In order to identify and isolate protein factors involved in initiation of DNA synthesis, specific chromatin configurations within the origin region will be first delineated by nuclease digestion studies; those chromatin complexes related to initiation of DNA synthesis will be identified by parallel replication studies in permeabilized cells. We will then use modified Western blot, band competition, and DNA cellulose affinity chromatography techniques to identify proteins in purified nuclear extracts that bind with high affinity and specificity to the DHFR origin region. Finally, we will use the nuclear protein extracts prepared for protein binding studies in complementation experiments in an effort to develop an in vitro assay for initiation of DNA synthesis in purified nuclei.
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