Distinguishing Specific and Nonspecific Complexes of Alkyladenine DNA Glycosylase

作者：Taylor Erin L; Kesavan Preethi M; Wolfe Abigail E; O'Brien Patrick J

来源：Biochemistry, 2018, 57(30): 4440-4454.

DOI：10.1021/acs.biochem.8b00531

Human alkyladenine DNA glycosylase (AAG) recognizes many alkylated and deaminated purine lesions and excises them to initiate the base excision DNA repair pathway. AAG employs facilitated diffusion to rapidly scan nonspecific sites and locate rare sites of damage. Nonspecific DNA binding interactions are critical to the efficiency of this search for damage, but little is known about the binding footprint or the affinity of AAG for nonspecific sites. We used biochemical and biophysical approaches to characterize the binding of AAG to both undamaged and damaged DNA. Although fluorescence anisotropy is routinely used to study DNA binding, we found unexpected complexities in the data for binding of AAG to DNA. Systematic comparison of different fluorescent labels and different lengths of DNA allowed binding models to be distinguished and demonstrated that AAG can bind with high affinity and high density to nonspecific DNA. Fluorescein-labeled DNA gave the most complex behavior but also showed the greatest potential to distinguish specific and nonspecific binding modes. We suggest a unified model that is expected to apply to many DNA binding proteins that exhibit affinity for nonspecific DNA. Although AAG strongly prefers to excise lesions from duplex DNA, nonspecific binding is comparable for single- and double-stranded nonspecific sites. The electrostatically driven binding of AAG to small DNA sites (similar to 5 nucleotides of single-stranded and similar to 6 base pairs of duplex) facilitates the search for DNA damage in chromosomal DNA, which is bound by nucleosomes and other proteins.

• 出版日期2018-7-31