This proposal is a Pilot Project (R21) that uses a novel approach to learn about the architecture of DNA sequences in chromosomes. The accessibility of DNA sequences along a chromosomes are probed in vivo by a novel assay involving recombination between homologous sequences in different chromosomal locations. The availability of the complete yeast genome has stimulated great interest in designing systematic approaches to the study of gene functions and gene-product interactions; however, less attention has been paid to non-coding aspects of the genome. Very little is yet understood about the way DNA sequences are arranged in chromosomes beyond their basic packaging with nucleosomes. The significance of periodic """"""""waves"""""""" of high and low GC content is not understood, nor is there much evidence of specific sequences that arrange chromosomes into putative loops or domains or of regions that ensure that the chromosomes are properly condensed at metaphase. To address these questions, a systematic study of the accessibility of regions of S. cerevisiae chromosomes will be undertaken. A competition assay has been devised, so that a double- strand break, introduced by the induction of the HO endonuclease, can either be repaired by gene conversion with an homologous donor locus (inserted at a test site), located on another chromosome or by a default pathway of single-strand annealing of homologous regions flanking the break. The ratio of inter-chromosomal gene conversions to the default pathway provides a measure of the ability of the donor region to pair and recombine an ectopic recipient site on another chromosome. A recombination cassette carrying the donor sequences will be inserted approximately every 10 kb along chromosomes I and XI and the ratio of the two repair events assessed at each point, to identify regions that are particularly accessible or inaccessible. Preliminary results suggest that there is at least 10 fold range in the ability of the donor sequence to participate in this type of repair. Two inter- chromosomal assays will be performed, by inducing the HO endonuclease in the G1 stage of the cell cycle, or in cells arrested at metaphase by nocodazole. In this way, regions that are particularly inaccessible in general and especially during chromosome condensation will be determined. These experiments will identify regions of chromosomes that are especially restricted or unusually accessible and provide the first insight into novel cis-acting elements that govern chromosome behavior.
