Elasticity-Driven Single Stranded Gap Creation Mechanism by an Exonuclease III/AP Endonuclease

Abstract

DNA damage occurs as many as 1 million individual molecular lesions per cell per day in human. One of the most frequently occurring DNA lesions is base damage, which are quickly processed into AP (apurinic/apyrimidinic) sites, resulting in “missing base” in DNA, so-called AP site. AP endonuclease catalyzes them for the Base Excision Repair (BER) process. Exonuclease III (exoIII) in E.coli functions as not only an AP endonuclease but also a 3′->5′ exonuclease exonuclease. We used single molecule FRET to examine how the multifunctional enzyme plays the dual role during the NER process. When the substrate is a blunt-ended DNA, the enzyme works as a distributive exonuclease, performing multiple rounds of binding and dissociation events during the cleavage reaction. However, in the presence of an AP site, exoIII cuts the AP site as an AP endonuclease, and then transforms into a processive exonuclease, performing a continuous digestion by strongly anchoring to the AP site. The strong binding affinity onto the AP site is the origin of the transformation mechanism, allowing the enzyme to digest a series of nucleotides without dissociation from the substrate. The close examination of the enzyme behavior reveals an elasticity-based mechanism where exoIII creates a gap by forming a single stranded intermediate loop during BER process. The size of the gap robustly relies on the physiological salt concentration due to the extent of the repulsive property in the negative charged singe stranded loop.