Compacting and organizing the meters of DNA in a human cell into the nucleus requires tight coiling into bead- like structures called nucleosomes. The nucleosomes further organize to form chromatin. Chromatin, like the DNA that surrounds it, contains information that is vital to maintaining proper gene activity. Thus, when a cell divides it is crucial for it to preserve both the sequence information contained in its DNA and the structural information in the surrounding nucleosomes. Many diseases result largely from changes in gene activity: defects in DNA replication and chromatin inheritance can lead to deleterious mutations and changes in gene expression that accelerate disease progression [28]. For example, it is hypothesized that the progression of many cancers relies on the preservation of chromatin markers over multiple cell divisions [29]. ! Despite the importance of these processes, the early steps of replication are poorly understood. In particular, compact nucleosomes must be disassembled ahead of the synthetic machinery for DNA replication to proceed, but little is known about this critical process [20]. The work outlined in this proposal will elucidate the mechanisms of chromatin unwinding during DNA replication. Specifically, it wil define the role of the replicative helicase, an enzyme that unwinds DNA, in nucleosome disassembly. Recent evidence suggests that some components of this helicase complex interact with chaperone proteins, helper proteins that bind to the core nucleosome proteins when they have dissociated from DNA [6, 13-18]. But the specific identification of a role for these proteins in nucleosome disasembly has been elusive: the chaperones have multiple functions that are intertwined with nucleosome disassembly [20]. In addition, nothing is known about the role of the full helicase complex in chromatin unwinding. Therefore, a primary goal of this proposal is to develop a novel assay using purified proteins that decouples nucleosome disassembly from other complicating cellular processes. This system will then be used to identify mutations that disrupt interactions between the helicase complex, chaperones, and core nucleosome proteins. These mutants will be a powerful reagent that will define the role of these proteins in nucleosome disassembly both in a purified system and in living cells. Specifically, these mutations should cause clear defects in DNA replication in living cells. Furthermore, they will also prevent nucleosome displacement during replication, and this can be determined by directly observing nucleosome positioning on DNA in living cells. Together, this work will provide significant insight into the mechanisms of the initiation of DNA replication and chromatin inheritance, two processes that are fundamental in our understanding of how human diseases arise.
This work will offer significant insight into the initiation of DNA replication and the inheritance of chromatin markers that influence gene expression: defects in either process can lead to disease. A thorough understanding of the mechanisms that control these processes will provide insight into how cells control their growth and how failures can cause disease to arise.
