These projects are designed to further our knowledge of chromatin replication. Histone synthesis and deposition are major aspects of this replication, as these proteins are required to package DNA such that it is accessible to effector molecules and polymerases. Studies on these processes have been initiated by synchronizing HTC cells and density labeling the DNA with iododeoxyuridine. These studies indicate that the synthesis and deposition of histones H3 and H4 is rigorously controlled, such that deposition occurs only on newly replicated DNA. When the limited synthesis of these histones occurs in G1, these histones are prevented from associating with DNA until the onset of DNA synthesis (S phase) and then specifically on the newly replicated DNA. Histones H2A and H2B deposit on either G1 DNA or S phase DNA at a rate suggestive of constant exchange. Whendeposition is occurring in S phase, H2A and H2B deposit on both newly replicated and nonreplicated DNA. Preliminary evidence suggests that the site for this non-replicated deposition is the DNA actively involved in RNA transcription. The histone variants X and Z appear to deposit primarily on nonreplicated DNA, as we cannot detect selective deposition of these histones on newly replicated DNA. The ubiquinated forms of new H2A, X, and Z are synthesized 4-fold greater in G1 versusS phase cells, which correlates well with the 4-fold decreased rate of deposition of new H2A, X, and Z in G1 cells. These results suggest that ubiquination occurs when these histones are released from the DNA and would suggest that ubiquinated forms of histones may be concentrated near active genes. We have initiated our studies on the thermodynamics of this exchange process (nucleosome stability) by utilizing RTG-1 cells which grow between 6~ and 25~C. We find that the in vivo exchange rate for H2A and H2B at 6~C is decreased to nondetectable levels, as manifested by the deposition of these histones only on newly replicated DNA. (K)