Characterization Of Mycobacterial Flap Endonuclease Fena And Rna Helicase Hely
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Uson, Maria Loressa
The genus Mycobacterium encompasses over 170 species, including the disease-causing Mycobacterium tuberculosis and its nonpathogenic relative Mycobacterium smegmatis. My thesis research focuses on mycobacterial proteins from two classes of enzymes critical for nucleic acid processing: helicases and nucleases. I purified and characterized FenA as a flap endonuclease and 5' exonuclease and HelY as an RNA helicase. Structure-specific DNA nucleases are found in all domains of life and play important roles in genome replication and maintenance, yet little is known about such nucleases in mycobacteria. I characterized M. smegmatis FenA as a manganese-dependent nucleic acid phosphodiesterase with flap endonuclease and 5’ exonuclease activities. FenA efficiently removes the 5’ App(dN) terminus of an aborted nick ligation reaction intermediate, which is formed under a variety of circumstances by bacterial ligases and especially by mycobacterial ligases D and C. We reported the 1.8 Å crystal structure of FenA, which assimilates three manganese ions in its active site. The three manganese ions are coordinated, directly and via waters, to a constellation of eight carboxylate side chains. I mutated each of the metal-coordinating residues individually to alanine and tested the mutants for flap endonuclease and 5’ exonuclease activities. I found that the carboxylate contacts to all three manganese ions are essential for FenA’s activities. A fourth manganese ion is engaged by a structure unique to FenA that comprises a 310 helix and a surface ?-loop. Mycobacteria possess a large ensemble of DNA helicases that function in DNA replication, repair and recombination. In comparison, little attention has been paid to the roster of RNA helicases in mycobacteria or their roles in RNA metabolism. We identified M. smegmatis HelY as a bacterial homolog of the eukaryotic Ski2 and Mtr4 helicases that regulate RNA 3' processing and turnover by the exosome. I showed that HelY is an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3'-to-5' helicase. HelY requires a 3' single-stranded RNA tail to displace the complementary strand of a RNA:DNA hybrid or RNA duplex. The biochemical properties of HelY are consistent with a role in mycobacterial RNA transactions, and we speculate that HelY functions in RNA catabolism.
Doctor of Philosophy
Attribution-NonCommercial-NoDerivatives 4.0 International
dissertation or thesis
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