Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes

Azide–alkyne cycloaddition (“click chemistry”) has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the “A-rule”, directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.     

Contribution of Microhomology to Genome Instability: Connection between DNA Repair and Replication Stress

Microhomology-mediated end joining (MMEJ) is a highly mutagenic pathway to repair double-strand breaks (DSBs). MMEJ was thought to be a backup pathway of homologous recombination (HR) and canonical nonhomologous end joining (C-NHEJ). However, it attracts more attention in cancer research due to its special function of microhomology in many different aspects of cancer. In particular, it is initiated with DNA end resection and upregulated in homologous recombination-deficient cancers. In this review, I summarize the following: (1) the recent findings and contributions of MMEJ to genome instability, including phenotypes relevant to MMEJ; (2) the interaction between MMEJ and other DNA repair pathways; (3) the proposed mechanistic model of MMEJ in DNA DSB repair and a new connection with microhomology-mediated break-induced replication (MMBIR); and (4) the potential clinical application by targeting MMEJ based on synthetic lethality for cancer therapy.