Characterization of the Searching Mechanism for a DNA Repair Enzyme.

DNA Repair;DNA Damage;Base Excision Repair;DNA Glycosylase;Linear Diffusion;Hopping;Sliding;Correlated Search;Biological Chemistry;Science;Chemical Biology

Hedglin, MarkWalter, Nils G. ;

University of Michigan

关键词: DNA Repair;    DNA Damage;    Base Excision Repair;    DNA Glycosylase;    Linear Diffusion;    Hopping;    Sliding;    Correlated Search;    Biological Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/78826/mhedgli_1.pdf?sequence=1&isAllowed=y

瑞士|英语

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

【 摘 要 】

Genomic DNA is constantly subjected to damaging modifications from reactive metabolites and environmental mutagens. The base excision repair pathway (BER) is responsible for repair of most modifications affecting single bases, collectively referred to as base lesions. Base lesions are the most frequently occurring type of DNA damage, with ~10,000 base lesions formed and repaired by BER in human cells everyday.However, in the context of the human genome, these base lesions are extremely rare, with only one of every 1.2 million nucleotides sustaining damage on any given day.The daunting task of locating single base lesions and initiating the BER pathway is bestowed upon DNA glycosylases, which catalyze removal of a wide variety of damaged nucleobases from DNA.We show that alkyladenine DNA glycosylase (AAG), a human protein that initiates repair of a diverse group of alkylated and deaminated purines, locates damage by a correlated searching mechanism whereby each binding encounter with DNA involves a search of multiple adjacent sites.This search is mediated by electrostatic binding interactions that allow linear diffusion along nonspecific DNA to locate target sites.We show that the N-terminus of AAG, which is dispensable for glycosylase activity, contributes to the correlated search by decreasing dissociation from DNA, possibly by directly contacting DNA.Furthermore, we demonstrate that AAG makes significant excursions from the surface of DNA while diffusing.Such events, referred to as hops, allow reorientation between strands, enabling AAG to search both strands of a DNA duplex in a single binding encounter.Hopping also allows AAG to bypass obstacles, such as tightly-bound proteins and helix-discontinuities.By comparing the behavior of AAG on oligonucleotides containing different lesions, we show that the efficiency of the search for damage depends on the identity of the base lesion.Whereas AAG recognizes the alkylated lesion, 1, N6-ethenoadenine with high efficiency, AAG requires multiple encounters with the oxidative lesion inosine. We infer that a highly redundant search allows multiple encounters with each lesion, ensuring that each lesion is repaired.Collectively, these studies provide insight into the molecular mechanism by which a DNA repair enzyme searches the genome for DNA damage.