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Originally discovered in a search for RNA polymerase II-associated factors, the Paf1 complex (Paf1C) is best characterized for its roles in regulating transcription elongation. The complex co-localizes with RNA polymerase II from the promoter to the 3’ end of genes and has been linked to a growing list of transcription-related processes including: elongation through chromatin, histone modifications, and recruitment of factors important in transcript maturation. The complex is conserved throughout eukaryotes and is comprised of the proteins Paf1, Ctr9, Cdc73, Rtf1, and Leo1. The domain structures of Paf1C subunits are largely undefined and have few clear homologs, making it difficult to postulate for or localize functions to the individual subunits. To understand mechanistically how Paf1C coordinates its functions and interactions, I took an approach utilizing biochemical, biophysical, and structural techniques to characterize proteins within Paf1C, specifically focusing on the Rtf1 subunit. The goal of my thesis work was to determine the molecular mechanism by which Rtf1 influences transcription and chromatin structure. To this end I focused on studying different functional domains within Rtf1. I provided a molecular description of how Rtf1 mediates Paf1C recruitment to elongating RNA polymerase II. Recruitment of Rtf1 is controlled by its centrally located Plus3 domain and a direct interaction with the conserved elongation factor Spt5. I solved the co-crystal structure of the human Plus3 domain bound to a phosphorylated C-terminal repeat of Spt5. The structure revealed the basis for recognition of the repeat motif of Spt5, an important component in the recruitment of regulatory factors to RNA polymerase II. I have performed further structural characterization of Rtf1, studying the N-terminal histone modification domain. To gain insight into the molecular mechanism underlying the domain’s function, I successfully crystallized and solved the structure of a minimal re

Originally discovered in a search for RNA polymerase II-associated factors, the Paf1 complex (Paf1C) is best characterized for its roles in regulating transcription elongation. The complex co-localizes with RNA polymerase II from the promoter to the 3’ end of genes and has been linked to a growing list of transcription-related processes including: elongation through chromatin, histone modifications, and recruitment of factors important in transcript maturation. The complex is conserved throughout eukaryotes and is comprised of the proteins Paf1, Ctr9, Cdc73, Rtf1, and Leo1. The domain structures of Paf1C subunits are largely undefined and have few clear homologs, making it difficult to postulate for or localize functions to the individual subunits. To understand mechanistically how Paf1C coordinates its functions and interactions, I took an approach utilizing biochemical, biophysical, and structural techniques to characterize proteins within Paf1C, specifically focusing on the Rtf1 subunit. The goal of my thesis work was to determine the molecular mechanism by which Rtf1 influences transcription and chromatin structure. To this end I focused on studying different functional domains within Rtf1. I provided a molecular description of how Rtf1 mediates Paf1C recruitment to elongating RNA polymerase II. Recruitment of Rtf1 is controlled by its centrally located Plus3 domain and a direct interaction with the conserved elongation factor Spt5. I solved the co-crystal structure of the human Plus3 domain bound to a phosphorylated C-terminal repeat of Spt5. The structure revealed the basis for recognition of the repeat motif of Spt5, an important component in the recruitment of regulatory factors to RNA polymerase II. I have performed further structural characterization of Rtf1, studying the N-terminal histone modification domain. To gain insight into the molecular mechanism underlying the domain’s function, I successfully crystallized and solved the structure of a minimal re

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2-Psy3 heterodimer preferentially binds synthetic forked DNA or 3′-DNA overhang substrates resembling structures used during HR in vivo . We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55-Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55-Rad57. Importantly, Csm2's interaction with Rad51 is dependent on Rad55, whereas Csm2's interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2-Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55-Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2-Psy3 heterodimer preferentially binds synthetic forked DNA or 3′-DNA overhang substrates resembling structures used during HR in vivo . We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55-Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55-Rad57. Importantly, Csm2's interaction with Rad51 is dependent on Rad55, whereas Csm2's interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2-Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55-Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.