The proposed research addresses two related questions: (1) What are the molecular mechanisms that regulate S-phase gene expression in Trypanosoma brucei? and (2) How is the expression of a specific mitochondrial DNA ligase regulated during the cell cycle and how does its expression contribute to kinetoplast structure and division? There appears to be little or no significant transcriptional regulation of protein coding genes in kinetoplastids. Rather, regulation of gene expression occurs largely at a post-transcriptional level. In earlier work with C. fasciculata we identified an octamer consensus sequence required for S-phase expression of several genes and also two protein complexes that bind to the octamer sequence with high specificity. Genes encoding these proteins are present in T. brucei as well as additional related genes that have not been investigated. We have successfully synchronized T. brucei and will use synchronized cultures to investigate S-phase gene expression and to identify and characterize the specific binding proteins. Subunits of these proteins will be identified using immunological methods and mass spectrometry. Protein complexes will also be purified and characterized using tandem affinity purification. RNA interference methods will be used to knock down expression of each of the binding proteins and to determine their importance in sequence-specific binding to the octamer sequence in transcripts. We will also use immunoprecipitation and RT-PCR to directly demonstrate the binding in vivo of these protein complexes to transcripts containing the octamer sequences. The C. fasciculata DNA ligase ka (LIG ka) is an unstable protein that shows cyclic expression and cyclic localization to the kinetoplast. We will examine the roles of octamer-related sequences in the cyclic expression of LIG ka and the adjacent LIG k¿ gene through point mutation of individual octamer-related sequences alone and in combination. These studies will address the possible effect of the cycling of each gene transcript on that of the other. We will also investigate the possible role of LIG ka in the regulated repair of gaps that remain at minicircle replication origins prior to their closure as a prelude to network division. The structure of the kDNA networks and of the network minicircles will be determined in T. brucei cells expressing LIG ka? at a constant and regulated level throughout the cell cycle. Since DNA ligases have been found to interact with DNA polymerases in gap repair reactions we will identify the specific mitochondrial DNA polymerase that might function together with LIG ka? in gap repair and will examine the ability of the ligase-polymerase complex to repair a model minicircle gap at the replication origin.