Mechanistic Studies of Alkylation Repair DNA Glycosylases.

Kinetics of DNA Repair Enzymes;AlkA and AAG Glycosylases;Biological Chemistry;Health Sciences;Science;Biological Chemistry

Taylor, ErinGoldstrohm, Aaron C ;

University of Michigan

关键词: Kinetics of DNA Repair Enzymes;    AlkA and AAG Glycosylases;    Biological Chemistry;    Health Sciences;    Science;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/120897/elmi_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Alkylating agents spontaneously react with nucleobases of DNA, and the resulting lesions have the propensity to be mutagenic and cytotoxic. The base excision repair pathway employs DNA glycosylases such as the E. coli AlkA and human AAG to find and initiate repair of alkylated bases. AlkA and AAG excise a wide range of alkylated lesions such as N-methylated purines and etheno adducts such as 1,N6-ethenoadenine (eA). This work characterizes AlkA and AAG to better understand the similarities and differences between these two structurally distinct enzymes that have independently evolved to repair a similar set of substrates. The kinetic and thermodynamic framework of AlkA was characterized with eA lesions and directly compared to the mechanism of AAG. In vitro kinetic assays show that AlkA binds rapidly and reversibly to eA prior to a slow excision step, and this binding is relatively unstable when compared to the more efficient AAG. As the expression of AlkA is induced in E. coli as part of an adaptive response to alkylation damage, this may compensate for lower efficiency.Several glycosylases have been reported to function as dimers but binding can also be complicated by specific and nonspecific binding modes. Therefore we characterized the stoichiometry and affinity of AAG for specific and nonspecific DNA binding. Fluorescence anisotropy measurements showed biphasic signals when AAG bound a 5′FAM-labeled eA substrate. However, utilization of eA fluorescence and EMSAs showed specific 1:1 complexes between AAG and the lesion site. The large changes in anisotropy and FAM fluorescence indicate that AAG binds DNA with high affinity near the FAM lesion site. We compared fluorescein and cyanine-like DyLight 647 dyes to demonstrate the importance of considering protein-fluorophore interactions when analyzing anisotropy data.Homologs of AlkA and AAG in Bacillus subtilis were also studied to ascertain their substrate specificities and necessity for alkylation repair. bAlkA was shown to be vital for the repair of cytotoxic lesions created by MMS, with bAAG providing backup repair activity. Both glycosylases exhibited an extended substrate range to include substrates canonical to the direct repair protein, AlkB, which is lacking in Bacillus subtilis.