We wish to understand basic principles that govern chromosome function, especially those involving spatial, temporal and functional coordination among different processes. Proposed research emphasizes synthetic comparisons among different organism and falls into four categories. First, several aspects of E.coli chromosome dynamics stemming from recent findings will be explored: three-dimensional disposition, mobility; cohesion and loss of cohesion between sister chromosomes (which occurs in a concerted process strikingly analogous to events in eukaryotic mitosis and has implications for the evolutionary development of chromosome segregation mechanisms in higher organisms); specialized behaviors of a peri-origin domain; and interlinkage of chromosome status and cell division. Second, we will further explore our finding that chromosome-based signal transduction by the yeast ATR homolog Mec1 mediates regulated progression of DNA replication through specific, genetically-defined """"""""slow zones"""""""". This process has implications for regulation of origin firing and the nature of DNA fragile sites. These processes are likely paradigmatic for events in higher eukaryotes. Genetic, physical and cytological studies are proposed. Third, we will continue ongoing studies of functional interplay between recombination and chromosome structure. One aspect of this work involves cytogenetic studies of meiosis in the filamentous fungus Sordaria macrospora. We will further investigate local recombination-mediated axis destabilization at the leptotene/zygotene transition, recombination-mediated juxtaposition of homolog axes via presynaptic alignment, roles of spindle checkpoint proteins in surveillance of prophase events and roles of selected cohesion and axis components in axis development. A second aspect will continue our recent physical studies of mitotic break-initiated recombination which, for the first, time, identify joint molecules involved in this process. Species and cell cycle variations will be further defined and requirements for biochemical and chromosome structure components will be probed by mutant studies. Fourth, we will consider mechanical properties of chromosomes by (i) developing ways to assess mechanical properties of HEAT repeat domains; (ii) exploring novel uses of magnetic forces to probe the mechanical properties of chromatin and whole chromosomes; and (iii) using our recently developed 3C analysis to explore local effects of double-strand breaks on chromatin state. ? ? ?
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