Abstract
Abstract The DEDD-family exonucleases are involved in various aspects of RNA processing and degradation, as well as DNA proofreading and repair. However, how this family of exonucleases binds at the 3'-end of a nucleic acid chain, and selects and digests their target substrates is mostly unknown. In this thesis, two DEDD-family exonucleases, CRN-4 and RNase T, have been selected for biochemical and structural studies to elucidate their functional roles in DNA degradation and RNA maturation, respectively. CRN-4 (Cell death-related nuclease 4) was identified as one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. We analyzed CRN-4’s biochemical properties and in vivo cellular functions, and determined the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4’s function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis. RNase T is a bacterial 3'-to-5' exonuclease involved in the final trimming of many stable RNA, including 5S and 23S ribosomal RNA and transfer RNA. The exonuclease activity of RNase T is blocked by a 3'-terminal cytosine and double-stranded structures. Our crystal structural analyses on four RNase T-DNA complexes show that a “C-filter” in RNase T screens out the nucleic acids with a 3'-terminal cytosine by inducing a disruptive conformational change at the active site. The two subunits of the RNase T dimer work together in binding a double-stranded structure, producing a minimum product of a duplex with a 2-nt or 1-nt 3' overhang, depending on the last base pair, G-C or U-A, in the duplex. Our results reveal the general principles and the underlying working mechanisms for the final trimming step made by RNase T in the maturation of ribosomal, transfer and small stable RNA. In summary, this thesis presents the biochemical properties and crystal structures of two DEDD-family nucleases. The structural studies in RNase T reveal the underlying working mechanism of this enzyme in substrate selection and digestion. As many DEDD nucleases share a fold and a conserved active site similar to that of CRN-4 and RNase T, the structural insight provided in this study not only shows the link between the DEDD domain arrangement and substrate specificity, but also reveals clues to understand the exonuclease activity and to identify possible substrates of other DEDD-family proteins, some of which have been linked directly to human diseases.