Molecular mechanism of bacteria and human AP endonucleases

Abstract

DNA damage occurs continuously by endogenous and environmental various factors such as ultraviolet light (UV), replication error and oxidative stress. More than 10,000 lesions of DNA are generated per cell per day. Therefore, DNA repair system is essential for genomic stability in living cells. In base excision repair (BER), damaged bases are rapidly converted to apurinic / apyrimidinic site (AP site) where base is missing. AP site is processed by AP endonuclease, incising phosphodiester bond 5′ to the AP site. AP endonuclease are highly conserved between bacteria and human. Many human diseases such as cancer and neurodegeneration are associated with mutations and post-translational modifications of AP endonuclease. This means that AP endonuclease can be a key component of development of therapeutics for various human diseases. In bacteria, exonuclease III (ExoIII) is a multi-functional enzyme which performs mainly both AP endonuclease and 3’-5’ exonuclease activities. In addition, both activities of ExoIII rely on only the single active site unlike other multifunctional enzymes. Unlike the bacterial AP endonuclease (ExoIII), in human cells, AP endonucleases have evolved into an AP endonuclease and 3′ to 5′ exonuclease. APE1 functions as a professional AP endonuclease but not 3’-5’ exonuclease, whereas APE2 mainly acts as a 3’-5’ exonuclease. However, despite a lot of researches have been done in an effort to understand the determinants of two activities of AP endonucleases, the fundamental mechanism of the difference between the AP endonuclease and exonuclease activities is still unclear due to the lack of enzyme kinetics and dynamics analyses. In order to understand molecular mechanisms of AP endonuclease and exonuclease, I introduced the single molecule fluorescence resonance energy transfer (smFRET) technique and a series of mutational analyses. In the first study, I aimed to decouple the multi-functionality of ExoIII into AP endonuclease and exonuclease. Next, in the second study, I aimed to recover the exonuclease function of hAPE1 using mutagenesis engineering. In ExoIII, I found that either W212 or F213 play role in recognizing AP sites on DNA, while the F213 play a critical role in not only the recognition of the AP site but also the stabilization of the melted 3′ terminal base leading to the catalytically competent state that facilitates the 3’ to 5′ exonuclease activity. During exonucleolytic cleavage, melting of the 3′ terminal nucleotide is a prerequisite for the catalysis. In addition, B-form helix of the duplex except the 3′ terminal base is stabilized by four phosphate-stabilizing residues (R90, Y109, K121 and N153) for exonuclease activity during 3’ terminal base to remain melted prior to cleavage. In hAPE1, I newly found that D70 and E126 which are electrostatically negative charges showed repulsive interaction with DNA, leading to reduce the affinity of DNA. In addition, enlarging the active site pocket by substituting F266 and W280 with alanine provides better interaction with W267, showing enhanced exonuclease activity. M270 hinder the translocation of hAPE1 to accommodate 3’ end nucleotide in the active site by intercalating into bases at single-strand and double-strand junction. Finally, I discovered that N229K stabilizes melted 3’ end nucleotide prior to cleavage. I successfully engineered hAPE1 to super-exonuclease (D70N, E126K, N229K, F266A, M270A, W280A) which exhibits ~50-fold higher exonuclease activity compared to the ExoIII and engineered hAPE1 shows new function which degradation of DNA in the processive manner, whereas ExoIII degrades DNA in the distributive manner. My works are expected to contribute to not only protein engineering fields for controlling biological enzymatic activities, but also development of drugs that target the AP endonuclease for cancer therapeutics. Moreover, because ExoIII is a widely used enzyme in many molecular biology assays (gene cloning, biosensor, DNA sequencing), the enhanced function of super exonucleases might be of interest in the field of biotechnology.