Our goals are to describe the chromatin structure in the amplified dihydrofolate reductase initiation locus. The first step that must occur during initiation of replication at an origin of replication is melting of the DNA duplex. We suggest that chromatin proteins could play a role in origin function in at least two ways: 1) by interacting directly with a cis-regulatory sequence(s) in the origin to effect helix melting, and 2) by governing the stability of the helix through long-range interactions that effect the stability of whole chromatin domains (possibly chromosomal loops).
Specific aims are: 1) to define the chromosomal domain structure in the neighborhood of the DHFR locus by performing nuclease sensitivity studies on a 500 kb region encompassing this locus; 2) to detect alterations in chromatin configuration near the initiation locus using nuclease hypersensitivity and methylation protection approaches in combination with high-resolution sequencing gels; 3) to analyze the nucleosomal arrangement in the initiation locus by a two-dimensional gel approach that maps the presence and/or abundance of the four histones on specific sequences; 4) to perform in vivo footprinting with nucleases and/or methylation protection on the initiation locus; and 5) to investigate the dependence of any altered chromosomal arrangements in the initiation locus on cell cycle position, using cells synchronized with a novel inhibitor that prevents initiation of replication.
