Structure and Function of Histone Chaperones

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DESCRIPTION (provided by applicant): The proper packaging of DNA helps prevent genetic damage and provides a platform for gene regulation. Thus, histones and their chaperones are required during replication and repair. More specialized chaperones play significant roles in oocytes, embryos and somatic cells, where they form histone storage complexes, decondense sperm chromatin and transfer histones onto DNA. To investigate the function of these specialized chaperones, we are doing a structural and biochemical survey of the Np and N1-families. Thus, we have shown that Np-family members form pentamers and decamers. In addition, Np, NO38, dNLP and NO29 each form supramolecular complexes when they are reconstituted with core histones. In our model these complexes contain a chaperone decamer and 5 histone octamers. When N1 is added to the Np-histone complex, a remarkable super-complex is formed. Thus, Np and N1 may function as co-chaperones to promote histone storage in oocytes, while Np transfers histones to sperm chromatin. Moreover, Np and N1 may pass stored histones to NAP1 and CAF1 or transfer histones directly onto DNA in the embryo. We are using X-ray crystallography and biochemical approaches to study the mechanism of histone binding by these chaperones. To this end, we will determine crystal structures for Np-family members complexed with histones and obtain crystal structures for NO29 and Mp62 in the ground state. In parallel experiments, we will characterize histone binding by N1 and its domains. We will then investigate the structures of N1 and N1-C alone and with their cognate histone complexes. Somatic members of the Np-family are nucleolar proteins (NO38, B23, ANO39 and Mp62) which may help remodel r-chromatin during ribosome biogenesis. These chaperones are targeted to the nucleolus by a C-terminal H-domain which binds to DNA. To understand this targeting process, we will characterize the DNA binding preferences of the NO38 H-domain and determine its structure.